U.S. patent application number 12/681511 was filed with the patent office on 2010-11-18 for therapeutic use of diaminophenothiazines.
This patent application is currently assigned to Wis Ta Laboratories Ltd.. Invention is credited to Charles Robert Harrington, John Mervyn David Storey, Claude Michel Wischik, Damon Jude Wischik.
Application Number | 20100290986 12/681511 |
Document ID | / |
Family ID | 40220337 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100290986 |
Kind Code |
A1 |
Wischik; Claude Michel ; et
al. |
November 18, 2010 |
THERAPEUTIC USE OF DIAMINOPHENOTHIAZINES
Abstract
The present invention relates generally to methods and materials
for use in the treatment or prophylaxis of diseases, for example
cognitive disorders, using diaminophenothiazines. In particular it
relates to treatments having optimised pharmacokinetic properties,
and dosage forms are intended to improve the relative cognitive or
CNS benefits of the diaminophenothiazines, for instance compared to
haematological effects.
Inventors: |
Wischik; Claude Michel;
(Aberdeen, GB) ; Wischik; Damon Jude; (London,
GB) ; Storey; John Mervyn David; (Old Aberdeen,
GB) ; Harrington; Charles Robert; (Aberdeen,
GB) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Wis Ta Laboratories Ltd.
|
Family ID: |
40220337 |
Appl. No.: |
12/681511 |
Filed: |
October 1, 2008 |
PCT Filed: |
October 1, 2008 |
PCT NO: |
PCT/GB08/03315 |
371 Date: |
April 2, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60960544 |
Oct 3, 2007 |
|
|
|
Current U.S.
Class: |
424/1.65 ;
424/9.1; 424/9.3; 424/9.6; 514/224.8 |
Current CPC
Class: |
A61P 25/16 20180101;
A61K 31/5415 20130101; A61P 25/28 20180101; A61K 45/06 20130101;
A61P 7/06 20180101; A61P 25/00 20180101; A61K 9/0065 20130101; A61K
31/542 20130101 |
Class at
Publication: |
424/1.65 ;
424/9.1; 424/9.3; 424/9.6; 514/224.8 |
International
Class: |
A61K 31/5415 20060101
A61K031/5415; A61K 51/04 20060101 A61K051/04; A61K 49/00 20060101
A61K049/00; A61P 25/28 20060101 A61P025/28; A61P 7/06 20060101
A61P007/06; A61P 25/16 20060101 A61P025/16 |
Claims
1. A method of treatment or prophylaxis of a cognitive or CNS
disorder in a patient, wherein said disorder is one which is
susceptible to treatment by a 3,7-diaminophenothiazine (DAPTZ)
compound, which method comprises orally administering to said
patient a dosage unit containing said DAPTZ compound in oxidised
form as active ingredient, wherein said dosage unit releases at
least 50% of said active ingredient within 30 minutes under
standard US/EU Pharmacopoeia dissolution conditions.
2. A method as claimed in claim 1 wherein the dosage unit enhances
the relative cognitive or CNS benefit vs. haematological effects of
the DAPTZ compound.
3. A method as claimed in claim 1 wherein the dosage unit enhances
absorption in the stomach and thereby minimises formation of DAPTZ
dimers favoured in the alkaline conditions of the small intestine
and lower gut.
4. A method as claimed in claim 1 wherein the dosage unit comprises
less than 120, 100, or 70 mg, and optionally 40-70 mg DAPTZ
compound and is administered 3/day or 4/day.
5. A method of treatment or prophylaxis of a cognitive or CNS
disorder in a patient, wherein said disorder is one which is
susceptible to treatment by a 3,7-diaminophenothiazine (DAPTZ)
compound, which method comprises orally administering to said
patient a dosage unit containing said DAPTZ compound in oxidised
form as active ingredient, wherein said dosage unit is
gastroretained.
6-8. (canceled)
9. A method of treatment or prophylaxis of a cognitive or CNS
disorder in a patient, wherein said disorder is one which is
susceptible to treatment by a 3,7-diaminophenothiazine (DAPTZ)
compound, which method comprises orally administering to said
patient a dosage unit containing said DAPTZ compound in stable
crystalline reduced form as active ingredient.
10-12. (canceled)
13. A method as claimed in claim 1 wherein the cognitive or CNS
disorder is a tauopathy and wherein the treatment of the tauopathy
is such that the DAPTZ compound causes inhibition of the
aggregation of the tau protein associated with said disease state
and also dissolution of tau aggregates in the brain of the patient
or subject.
14. (canceled)
15. A method as claimed in claim 13 wherein the cognitive or CNS
disorder is selected from the list consisting of: Alzheimer's
disease, Pick's disease, Progressive Supranuclear Palsy (PSP),
fronto-temporal dementia, parkinsonism linked to chromosome 17,
disinhibition-dementia-parkinsonism-amyotrophy complex,
pallido-ponto-nigral degeneration, Guam-ALS syndrome;
pallido-nigro-luysian degeneration, cortico-basal degeneration and
mild cognitive impairment.
16. (canceled)
17. A method as claimed in claim 1 wherein the cognitive or CNS
disorder is a synucleinopathy, which is selected from the list
consisting of: Parkinson's Disease, dementia with Lewy bodies,
multiple system atrophy, drug-induced parkinsonism, and pure
autonomic failure (PAF).
18. A method as claimed in claim 1 wherein the patient is one who
is believed to be at above average risk of a haematological
disorder, the effects of which may otherwise be exacerbated by the
DAPTZ compound.
19. A method as claimed in claim 18 wherein the patient is one
known or believed to be suffering from a haemoglobinopathy which is
optionally Sickle-cell disease, thalassemia, methaemoglobinemia; an
anemia which is optionally a haemolytic anemia; a haematological
malignancy which is optionally lymphoma, myeloma, plasmacytoma or
leukemia, a coagulopathy which is optionally hemophilia.
20. A method as claimed in claim 1 wherein the dosage unit is
provided as a pharmaceutical composition comprising the DAPTZ
compound and a pharmaceutically acceptable carrier, diluent, or
excipient.
21. A method as claimed in claim 1 wherein the pharmaceutical
composition is for a combination therapy and comprises in addition
to the DAPTZ compound a further active ingredient selected from: a
cholinesterase inhibitor; an NMDA receptor antagonist; a muscarinic
receptor agonist; and an inhibitor of conversion of amyloid
precursor protein to beta-amyloid.
22. A method as claimed in claim 1 wherein said DAPTZ compound is
selected from compounds of the following formulae and
pharmaceutically acceptable salts, mixed salts, solvates, and
hydrates thereof: ##STR00053## wherein each one of R.sup.1,
R.sup.2, R.sup.4, R.sup.6, R.sup.8, and R.sup.9 is independently
selected from: --H; --F; --Cl; --Br; --I; --OH; --OR; --SH; --SR;
--NO.sub.2; --C(.dbd.O)R; --C(.dbd.O)OH; --C(.dbd.O)OR;
--C(.dbd.O)NH.sub.2; --C(.dbd.O)NHR; --C(.dbd.O)NR.sub.2;
--C(.dbd.O)NR.sup.N1R.sup.N2; --NH.sub.2; --NHR; --NR.sub.2;
--NR.sup.N1R.sup.N2; --NHC(.dbd.O)H; --NRC(.dbd.O)H;
--NHC(.dbd.O)R; --NRC(.dbd.O)R; --R; wherein each R is
independently selected from: unsubstituted aliphatic
C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl; unsubstituted
aliphatic C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
unsubstituted C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl;
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; wherein, in each group
--NR.sup.N1R.sup.N2, independently, R.sup.N1 and R.sup.N2 taken
together with the nitrogen atom to which they are attached form a
ring having from 3 to 7 ring atoms; and wherein, in each group
--NR.sup.3NAR.sup.3NA, if present: each one of R.sup.3NA and
R.sup.3NB is independently selected from: --H; unsubstituted
aliphatic C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl;
unsubstituted aliphatic C.sub.2-6alkenyl; substituted aliphatic
C.sub.2-6alkenyl; unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl; unsubstituted C.sub.6-10carboaryl; substituted
C.sub.6-10carboaryl; unsubstituted C.sub.5-10heteroaryl;
substituted C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; or: R.sup.3NA and R.sup.3NB
taken together with the nitrogen atom to which they are attached
form a ring having from 3 to 7 ring atoms; and wherein, in each
group .dbd.NR.sup.3NC, if present, R.sup.3NC is independently
selected from: --H; unsubstituted aliphatic C.sub.1-6alkyl;
substituted aliphatic C.sub.1-6alkyl; unsubstituted aliphatic
C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
unsubstituted C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl;
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; and wherein, in each group
--NR.sup.7NAR.sup.7NA, if present: each one of R.sup.7NA and
R.sup.7NB is independently selected from: --H; unsubstituted
aliphatic C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl;
unsubstituted aliphatic C.sub.2-6alkenyl; substituted aliphatic
C.sub.2-6alkenyl; unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl; unsubstituted C.sub.6-10carboaryl; substituted
C.sub.6-10carboaryl; unsubstituted C.sub.5-10heteroaryl;
substituted C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; or: R.sup.7NA and R.sup.7NB
taken together with the nitrogen atom to which they are attached
form a ring having from 3 to 7 ring atoms; and wherein, in each
group .dbd.NR.sup.7NC, if present, R.sup.7NC is independently
selected from: --H; unsubstituted aliphatic C.sub.1-6alkyl;
substituted aliphatic C.sub.1-6alkyl; unsubstituted aliphatic
C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
unsubstituted C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl;
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; and wherein X.sup.-, if
present, is one or more anionic counter ions to achieve electrical
neutrality.
23. A method as claimed in claim 22, wherein each one of R.sup.1,
R.sup.2, R.sup.4, R.sup.6, R.sup.8, and R.sup.9 is independently
selected from: --H; --R.
24. (canceled)
25. A method as claimed in claim 23, wherein substituents on R, if
present, are independently selected from: --F; --Cl; --Br; --I;
--OH; --OR; --C(.dbd.O)OH; --C(.dbd.O)OR'; --R', wherein each R' is
independently selected from: unsubstituted aliphatic
C.sub.1-6alkyl; unsubstituted aliphatic C.sub.2-6alkenyl;
unsubstituted C.sub.3-6cycloalkyl; unsubstituted
C.sub.6-10carboaryl; unsubstituted C.sub.5-10heteroaryl; and
unsubstituted C.sub.6-10carboaryl-C.sub.1-4alkyl.
26. (canceled)
27. A method as claimed in claim 22, wherein each one of R.sup.1,
R.sup.2, R.sup.4, R.sup.6, R.sup.8, and R.sup.9 is independently
selected from: --H, -Me, -Et, -nPr, and -iPr.
28. (canceled)
29. A method as claimed in claim 22, wherein, in each group
--NR.sup.3NAR.sup.3NB and --NR.sup.7NAR.sup.7NB, if present, each
one of R.sup.3NA and R.sup.3NB and --NR.sup.7NA and R.sup.7NB, is
independently selected from: --H, -Me, -Et, -nPr, and -iPr.
30. A method as claimed in claim 22, wherein, in each group
.dbd.NR.sup.3NC and in each group .dbd.NR.sup.7NC, if present,
.dbd.R.sup.3NC and .dbd.NR.sup.7NC are independently selected from:
--H, -Me, -Et, -nPr, and -iPr.
31. (canceled)
32. A method as claimed in claim 22, wherein X.sup.-, if present,
is one or more anionic counter ions to achieve electrical
neutrality, optionally selected from Cl.sup.-, Br.sup.-, I.sup.-,
or NO.sub.3.sup.-.
33. A method as claimed in claim 1 wherein the DAPTZ compound is
selected from the following compound, and pharmaceutically
acceptable salts, mixed salts, hydrates, and solvates thereof:
##STR00054##
34. A method as claimed in claim 9 wherein said DAPTZ compound in
stable crystalline reduced form is selected from compounds of the
following formula and pharmaceutically acceptable salts, solvates,
and hydrates thereof: ##STR00055## wherein: each of R.sup.1 and
R.sup.9 is independently selected from: --H, C.sub.2-4alkenyl, and
halogenated C.sub.1-4alkyl; each of R.sup.3NA and R.sup.3NB is
independently selected from: --H, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl; each of R.sup.7NA and R.sup.7NB is independently
selected from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and
halogenated C.sub.1-4alkyl; each of HX.sup.1 and HX.sup.2 is
independently a protic acid.
35. A method as claimed in claim 34, wherein each of R.sup.1 and
R.sup.9 is independently --H.
36-40. (canceled)
41. A method as claimed in claim 34, wherein each of HX.sup.1 and
HX.sup.2 is independently selected from HCl, HBr, and HI.
42-45. (canceled)
46. A drug product comprising a dosage unit as defined in claim 1
wherein the dosage unit comprises at least one pharmaceutically
acceptable excipient and, as an active ingredient, an isolated pure
DAPTZ compound, which dosage unit enhances the relative cognitive
or CNS benefit vs. haematological effects of the DAPTZ compound by
enhancing absorption in the stomach and thereby minimising
formation of DAPTZ dimers favoured in the alkaline conditions of
the small intestine and lower gut.
47. A method of labelling an aggregated disease protein associated
with a neurodegenerative disorder in the brain of a patient,
wherein said aggregated disease protein is one which is susceptible
to labelling by a DAPTZ compound, which method comprises orally
administering to said patient a dosage unit containing said DAPTZ
compound in oxidised form as active-labelled ingredient, wherein
said dosage unit releases at least 50% of said active ingredient
within 30 minutes under standard conditions.
48. A method of labelling an aggregated disease protein associated
with a neurodegenerative disorder in the brain of a patient,
wherein said aggregated disease protein is one which is susceptible
to labelling by a DAPTZ compound, which method comprises orally
administering to said patient a dosage unit containing said DAPTZ
compound in oxidised form as active-labelled ingredient, wherein
said dosage unit is gastroretained.
49. A method of labelling an aggregated disease protein associated
with a neurodegenerative disorder in the brain of a patient,
wherein said aggregated disease protein is one which is susceptible
to labelling by a DAPTZ compound, which method comprises orally
administering to said patient a dosage unit containing said DAPTZ
compound in stable crystalline reduced form as active-labelled
ingredient.
50. A method as claimed in claim 47 wherein the DAPTZ compound is
selected from compounds of the following formulae and
pharmaceutically acceptable salts, mixed salts, solvates, and
hydrates thereof: ##STR00056## wherein each one of R.sup.1,
R.sup.2, R.sup.4, R.sup.6, R.sup.8, and R.sup.9 is independently
selected from: --H; --F; --Cl; --Br; --I; --OH; --OR; --SH; --SR;
--NO.sub.2; --C(.dbd.O)R; --C(.dbd.O)OH; --C(.dbd.O)OR;
--C(.dbd.)NH.sub.2; --C(.dbd.O)NHR; --C.dbd.(O)NR.sub.2;
--C(.dbd.O)NR.sup.N1R.sup.N2; --NH.sub.2; --NHR; --NR.sub.2:
--NR.sup.N1R.sup.N2; --NHC(.dbd.O)H; --NRC(.dbd.O)H;
--NHC(.dbd.O)R; --NRC(.dbd.O)R; --R. wherein each R is
independently selected from: unsubstituted aliphatic
C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl; unsubstituted
aliphatic C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
unsubstituted C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl;
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; wherein, in each group
--NR.sup.N1R.sup.N2, independently, R.sup.N1 and R.sup.N2 taken
together with the nitrogen atom to which they are attached form a
ring having from 3 to 7 ring atoms; and wherein, in each group
--NR.sup.3NAR.sup.3NA, if present: each one of R.sup.3NA and
R.sup.3NB is independently selected from: --H; unsubstituted
aliphatic C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl;
unsubstituted aliphatic C.sub.2-6alkenyl substituted aliphatic
C.sub.2-6alkenyl; unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl; unsubstituted C.sub.6-10carboaryl; substituted
C.sub.6-10carboaryl; unsubstituted C.sub.5-10heteroaryl;
substituted C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; or: R.sup.3NA and R.sup.3NB
taken together with the nitrogen atom to which they are attached
form a ring having from 3 to 7 ring atoms; and wherein, in each
group .dbd.NR.sup.3NC, if present, R.sup.3NC is independently
selected from: --H; unsubstituted aliphatic C.sub.1-6alkyl;
substituted aliphatic C.sub.1-6alkyl; unsubstituted aliphatic
C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
unsubstituted C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl;
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; and wherein, in each group
--NR.sup.7NAR.sup.7NA, if present: each one of R.sup.7NA and
R.sup.7NB is independently selected from: --H; unsubstituted
aliphatic C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl;
unsubstituted aliphatic C.sub.2-6alkenyl; substituted aliphatic
C.sub.2-6alkenyl; unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl; unsubstituted C.sub.6-10carboaryl; substituted
C.sub.6-10carboaryl; unsubstituted C.sub.5-10heteroaryl;
substituted C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; or: R.sup.7NA and R.sup.7NB
taken together with the nitrogen atom to which they are attached
form a ring having from 3 to 7 ring atoms; and wherein, in each
group .dbd.NR.sup.7NC, if present, R.sup.7NC is independently
selected from: --H; unsubstituted aliphatic C.sub.1-6alkyl;
substituted aliphatic C.sub.1-6alkyl; unsubstituted aliphatic
C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
unsubstituted C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl;
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; and wherein X.sup.-, if
present, is one or more anionic counter ions to achieve electrical
neutrality.
51. A method as claimed in claim 47 wherein the DAPTZ compound
incorporates, is conjugated to, is chelated with, or is otherwise
associated with, one or more detectable labels optionally selected
from: isotopes, radioisotopes, positron-emitting atoms, magnetic
resonance labels, dyes, fluorescent markers, antigenic groups, or
therapeutic moieties.
52. A method as claimed in claim 47 wherein the DAPTZ compound
incorporates a positron-emitting atom.
53-55. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates generally to methods and
materials for use in the treatment or prophylaxis of diseases, for
example cognitive disorders, using diaminophenothiazines. In
particular it relates to treatments having optimised
pharmacokinetic properties.
BACKGROUND ART
[0002] 3,7-diaminophenothiazine (DAPTZ) compounds have previously
been shown to inhibit tau protein aggregation and to disrupt the
structure of PHFs, and reverse the proteolytic stability of the PHF
core (see WO96/30766, F Hoffman-La Roche). Such compounds were
disclosed for use in the treatment and prophylaxis of various
diseases, including AD and Lewy Body Disease, and included
methylthioninium chloride ("MTC").
[0003] WO96/30766 describes, in the case of oral administration, a
daily dosage of about 50 mg to about 700 mg, preferably about 150
mg to about 300 mg, divided in preferably 1-3 unit doses.
[0004] Other disclosures of phenothiazines in the area of
neurodegenerative disorders include WO 02/075318, WO
2005/030676.
[0005] It was known in the art that DAPTZ compounds can occur in a
charged (oxidised) form and an uncharged (reduced or "leuko") form.
It was also known that the cellular absorption of these differed.
Additionally, it was known that such compounds could in principle
have adverse haematological effects and other side effects at
certain doses.
[0006] WO 02/055720 (The University Court of the University of
Aberdeen) discusses the use of reduced forms of
diaminophenothiazines specifically for the treatment of a variety
of protein aggregating diseases, although the disclosure is
primarily concerned with tauopathies. WO 02/055720 discusses a
preliminary pharmacokinetic model based on studies of urinary
excretion data sets in humans, dogs and rats by DiSanto and Wagner,
J Pharm Sci 1972, 61:1086-1090 and 1972, 61:1090-1094 and Moody et
al., Biol Psych 1989, 26: 847-858. It further notes that the only
form of methylene blue which crosses the blood-brain barrier after
iv administration is the reduced form. Based on in vitro activity
for the reduced forms of diaminophenothiazines therein, a suggested
daily dosage was 3.2-3.5 mg/kg, and dosages of 20 mg tds, 50 mg tds
or 100 mg tds, combined with 2.times.mg ratio of ascorbic acid in
such a manner as to achieve more than 90% reduction prior to
ingestion were also described.
[0007] However WO 02/055720 did not provide a model which
integrated blood level data such as that described by Peter et al.
(2000) Eur J Clin Pharmacol 56: 247-250 or provide a model
validated by clinical trial data. Indeed, as described below, the
Peter et al. data contradicted the earlier data from DiSanto and
Wagner as regards terminal elimination half-life.
[0008] May et al. (Am J Physiol Cell Physiol, 2004, Vol. 286, pp.
C1390-C1398) showed that human erythrocytes sequentially reduce and
take up MTC i.e. that MTC itself is not taken up by the cells but
rather that it is the reduced from of MTC that crosses the cell
membrane. They also showed that the rate of uptake is enzyme
dependent; and that both MTC and reduced MTC are concentrated in
cells (reduced MTC re-equilibrates once inside the cell to form
MTC).
[0009] Nevertheless, the optimisation of an appropriate therapeutic
dose of DAPTZ compounds such as MTC, and their formulation, in
particular to optimise desired activity or minimise adverse side
affects are complex problems. A major barrier to this is the lack
of a suitable pharmacokinetic model. Thus it can be seen that the
provision of such a model, and hence teaching about addressing one
or more of these problems, would provide a contribution to the
art.
[0010] Prior filed, unpublished, application PCT/GB2007/001103
discloses compounds including:
##STR00001##
[0011] These compounds may be considered to be a stabilized reduced
form by comparison with, for example, MTC.
[0012] PCT/GB2007/001103 describes dosage units comprising 20 to
300 mg of the DAPTZ compounds described therein e.g. 30 to 200 mg,
for example 30 mg, 60 mg, 100 mg, 150 mg, 200 mg. A suitable dose
of the DAPTZ compound is suggested in the range of about 100 ng to
about 25 mg (more typically about 1 .mu.g to about 10 mg) per
kilogram body weight of the subject per day e.g. 100 mg, 3 times
daily, 150 mg, 2 times daily, 200 mg, 2 times daily.
DISCLOSURE OF THE INVENTION
[0013] Methylthioninium chloride ("MTC") is the active ingredient
of a proprietary therapeutic preparation (designated "Rember.TM.")
being developed for the treatment of AD and related dementias. A
clinical trial has been conducted in which therapeutic efficacy has
been demonstrated over 50 weeks of treatment in mild and moderate
AD.
[0014] Utilising the results of this trial, the present inventors
have developed a completely novel integrated pharmacokinetic model
applicable to the human oral dosage of DAPTZ compounds including,
but not limited to, MTC. The model has major implications for
defining the parameters that determine optimal oral dosing in terms
of safety and efficacy, and implies novel treatment modalities for
the treatment of cognitive disorders. The new model is shown to be
accurate in that it predicts urinary excretion, and correctly
predicts kinetics of the brain tissue compartment verified by the
pig study.
[0015] Briefly, the clinical trial showed that MTC has two systemic
pharmacological actions: cognitive effects and haematological
effects, but that unexpectedly these actions are separable.
Specifically the cognitive effects do not show a monotonic
dose-response relationship, whereas the haematological effects do.
The inventors propose that two distinct species are responsible for
the two types of pharmacological activity: MTC absorbed as the
uncharged Leuco-MT form being responsible for the beneficial
cognitive activity, and MTC absorbed as an oxidised dimeric species
being responsible for the oxidation of haemoglobin. Since these
effects are mechanistically distinct, they may be separately
manipulated such as to maximising the bioavailability of the
therapeutically active (cognitively effective) species.
[0016] Thus these findings have profound implications for the
dosing of both oxidised and leuco-DAPTZ compounds, in each case
such as to maximise therapeutic activity and therefore reducing
side effects by optimisation of dosing regime and formulation
relevant to the agent in question.
Oxidised DAPTZ Compounds--Rapid Dissolution Forms
[0017] As can be seen from FIG. 31A, there is a steep loss of
predicted efficacy as the observed percentage capsule dissolution
at 30 minutes drops below 20%. This confirms that rapid dissolution
is critical for therapeutic activity and can be explained by the
critical role of the stomach in the absorption of the
Methylthioninium (MT)-moiety in its therapeutically active
form.
[0018] Specifically, according to the delayed dissolution
hypothesis, a quite distinct form of MT is responsible for
haematological side effects. This was postulated to be a dimer, the
formation of which is favoured in the alkaline conditions of the
small intestine and lower gut. Therefore, the haematological side
effects observed in the clinical trial were a specific consequence
of the gelatine capsule formulation used in the study (and in
particular its rate of dissolution--see FIG. 7) rather than an
inherent feature of the MT moiety itself, if absorbed via the
stomach.
[0019] Therefore, in the design of an improved formulation of MTC
or other DAPTZ compounds, the attainment of predicted efficacy is
critically determined by the requirement that the dissolution of
the investigational medicinal product (i.e. tablet or capsule) be
greater than 50% in 30 minutes in standard conditions.
[0020] Thus in one aspect there is disclosed a method of treatment
of a cognitive or CNS disorder in a patient, wherein said disorder
is one which is susceptible to treatment by a DAPTZ compound,
[0021] which method comprises orally administering to said patient
a dosage unit containing said DAPTZ compound in oxidised form as
active ingredient, [0022] wherein said dosage unit releases at
least 50% of said active ingredient within 30 minutes under
standard conditions.
[0023] The treatment of the cognitive or CNS disorder will be such
as to maximise the relative cognitive or CNS benefit vs.
haematological effects of the DAPTZ compound (see e.g. FIG. 7).
Capsule dissolution is determined by the amount of DAPTZ released
into the aqueous phase of simulated gastric fluid (SGF) under
standard US/EU Pharmacopoeia dissolution conditions. This is
described in Example 11.
[0024] Dosage units of this form will therefore maximise absorption
in the stomach, and more critically minimise formation of dimers
which is favoured in the alkaline conditions of the small intestine
and lower gut.
[0025] Preferably greater than 95%, 90%, 85%, 80%, 75%, 70%, 60% or
50% will be absorbed by the stomach in less than 30 minutes.
[0026] Formulations and delivery vehicles suitable for this rapid
dissolution are discussed in more detail below.
[0027] The amount of oxidised DAPTZ in the dosage form will be a
therapeutically-effective amount. However based on the disclosure
herein it can be seen that very high doses (where dissolution is
delayed) will lead to only limited absorption of the nominal dose
in the stomach via the reductase mechanism leading to undesirable
delayed absorption from the small intestine at higher pH via
formation of dimers.
[0028] Thus preferably the dosage unit comprises less than 120 mg,
less than 100, less than 70, most preferably from 40-70 mg (e.g.
40, 45, 50, 55, 60, 65, or 70) and is administered 3/day or 4/day
(see e.g. FIGS. 29 & 30 & 32 & 36).
Oxidised DAPTZ Compounds--Gastric Retention Forms
[0029] Thus in one aspect there is disclosed a method of treatment
of a cognitive or CNS disorder in a patient, wherein said disorder
is one which is susceptible to treatment by a DAPTZ compound,
[0030] which method comprises orally administering to said patient
a dosage unit containing said DAPTZ compound in oxidised form as
active ingredient, [0031] wherein said dosage unit is
gastroretained.
[0032] The treatment of the cognitive or CNS disorder will be such
as to maximise the relative cognitive or CNS benefit vs.
haematological effects of the DAPTZ compound (see e.g. FIG. 7).
[0033] A gastroretained form will preferably be held in the stomach
for at least 30 minutes, more preferably at least 1, 2, 3, 4, 5, 6,
8, 12 hours or more.
[0034] Formulations and delivery vehicles suitable for
gastroretention are discussed in more detail below.
[0035] The amount of oxidised DAPTZ in the gastroretained dosage
form will be a therapeutically-effective amount. By minimising
transit to the small intestine (and hence formation of
therapeutically inactive dimers) higher loadings of oxidised DAPTZ
are feasible. Thus preferably the dosage unit comprises at least
50, 60, 70, 80, 90 or 100 mg, or more e.g. 200, 300, 400, 500
mg.
Reduced DAPTZ Compounds
[0036] The relationships described herein have implications as
regards the conventional approach to achieving using a more
convenient dosing regime, ie 2/day or 1/day. These dosing regimes
are in principle more desirable in patients with dementia, who are
forgetful and hence need prompting to take medication. The
conventional approach to achieving a more convenient dosing regime
is to create a slow-release formulation. However, the present
analysis indicates that, on the contrary, a standard slow-release
formulation of an oxidised DAPTZ form of a therapeutic product
would essentially eliminate efficacy, as illustrated conveniently
by the properties of the 100 mg capsule in TRx-014-001 in the
Examples hereinafter.
[0037] Thus, for the reasons discussed above, it would not be
feasible to generate a delayed-release formulation of an oxidised
DAPTZ-based medicinal product. However, this would not be the case
for drug products where the DAPTZ compound is in reduced form. This
is because the leuco-forms of such compounds cannot dimerise, since
they are not `flat` molecule do not have the charge which permits
stabilisation of the dimeric form by charge neutralisation.
[0038] Thus in a further aspect there is disclosed a method of
treatment of a cognitive or CNS disorder in a patient, wherein said
disorder is one which is susceptible to treatment by a DAPTZ
compound, [0039] which method comprises orally administering to
said patient a dosage unit containing said DAPTZ compound in stable
crystalline reduced form as active ingredient.
[0040] As described below preferably the compound is such as to
treat the cognitive or CNS disorder and to maximise the relative
cognitive or CNS benefits vs. haematological effects of the DAPTZ
compound.
[0041] In particular, based on the teaching herein, loss due to
initial non-absorption from the stomach would be greatly reduced
since the stably reduced crystalline forms have greater solubility
than the oxidised equivalents and would not require the activity of
the thiazine-dye reductase (May et al., 2004) which is presumed to
exist in the stomach and is presumed to be necessary for absorption
(see predicted absorption in FIG. 33).
[0042] It is therefore inferred that substantially higher efficacy
and superior dosing regime could be achieved using the L-MTx form
of the methylthioninium moiety. The amount of reduced DAPTZ in the
dosage form will be a therapeutically-effective amount.
[0043] In particular a delayed-release formulation (e.g. 1/day) of
the reduced DAPTZ compound at between 100-1000 mg would in
principle not lead to the adverse consequences of delayed
absorption (see FIG. 38). Thus in the light of the disclosure
herein dosages of up to 1000 mg or more (e.g. 100, 200, 300, 400,
500, 600, 700, 800, 900 or 1000 mg) given 1/day or more may be
considered.
[0044] Preferably this is a slow or delayed release formulation
i.e. release of less than <50% in 1 hour, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours.
[0045] As can be seen from FIGS. 34 & 35, it is predicted that
a level of efficacy of -8.1 ADAS-cog units could be achieved on a
dosing regime of 100 mg of the reduced DAPTZ (described as "L-MTx
form") administered twice daily. This could also be achieved by
dosing with 60 mg 3 times per day. Even higher efficacy levels
would be expected using 100 mg or higher administered 3 times per
day.
[0046] The preferred reduced DAPTZ compounds of the present
invention may conveniently be described as being in a "stabilized
crystalline reduced form" and are described in prior filed,
unpublished, application PCT/GB2007/001103. It will appreciated,
however, that even these compounds may autoxidize to some extent to
give the corresponding oxidized forms. Thus, it is likely, if not
inevitable, that compositions comprising the stabilized crystalline
reduced form DAPTZ compounds of the present invention will contain,
as an impurity, as least some of the corresponding oxidized
compound.
[0047] In aspects of the present invention pertaining to these
stabilized crystalline reduced form DAPTZ compounds, these oxidised
DAPTZ compounds may represent no more than 50% by weight, e.g., no
more than 40% by weight, e.g., no more than 30% by weight,
preferably e.g., no more than 20% by weight, e.g., no more than 10%
by weight, e.g., no more than 5% by weight, e.g., no more than 3%
by weight, e.g., no more than 2% by weight, e.g., no more than 1%
by weight of the total DAPTZ content of the dosage form.
Treatment
[0048] The term "treatment," as used herein in the context of
treating a condition, pertains generally to treatment and therapy
of a human, in which some desired therapeutic effect is achieved,
for example, the inhibition of the progress of the condition, and
includes a reduction in the rate of progress, a halt in the rate of
progress, regression of the condition, amelioration of the
condition, and cure of the condition.
[0049] The present invention further includes prophylactic measures
(i.e., prophylaxis, prevention).
[0050] The term "therapeutically-effective amount," as used herein,
pertains to that amount of an active compound, or a material,
composition or dosage from comprising an active compound, which is
effective for producing some desired therapeutic effect,
commensurate with a reasonable benefit/risk ratio, when
administered in accordance with a desired treatment regimen.
[0051] Similarly, the term "prophylactically-effective amount," as
used herein, pertains to that amount of an active compound, or a
material, composition or dosage from comprising an active compound,
which is effective for producing some desired prophylactic effect,
commensurate with a reasonable benefit/risk ratio, when
administered in accordance with a desired treatment regimen.
[0052] The term "treatment" includes combination treatments and
therapies, in which two or more treatments or therapies are
combined, for example, sequentially or simultaneously. Combination
treatments are discussed in more detail hereinafter.
Cognitive or CNS Disorders
[0053] Preferred cognitive or CNS disorders are described below.
Further neuro-degenerative disorders are described in the Examples
hereinafter.
[0054] The cognitive disorder may be a tauopathy condition in a
patient (see e.g. WO96/30766). As well as Alzheimer's disease (AD),
the pathogenesis of neurodegenerative disorders such as Pick's
disease and Progressive Supranuclear Palsy (PSP) appears to
correlate with an accumulation of pathological truncated tau
aggregates in the dentate gyrus and stellate pyramidal cells of the
neocortex, respectively. Other dementias include fronto-temporal
dementia (FTD); parkinsonism linked to chromosome 17 (FTDP-17);
disinhibition-dementia-parkinsonism-amyotrophy complex (DDPAC);
pallido-ponto-nigral degeneration (PPND); Guam-ALS syndrome;
pallido-nigro-luysian degeneration (PNLD); cortico-basal
degeneration (CBD) and others (see Wischik et al. 2000, loc. cit,
for detailed discussion--especially Table 5.1). All of these
diseases, which are characterized primarily or partially by
abnormal tau aggregation, are referred to herein as
"tauopathies".
[0055] In this and all other aspects of the invention relating to
tauopathies, preferably the tauopathy is selected from the list
consisting of the indications above, i.e., AD, Pick's disease, PSP,
FTD, FTDP-17, DDPAC, PPND, Guam-ALS syndrome, PNLD, and CBD.
[0056] In one preferred embodiment the tauopathy is Alzheimer's
disease (AD).
[0057] Where the disease is any tauopathy, the method of treatment
of the tauopathy may be such that the DAPTZ compound causes
inhibition of the aggregation of the tau protein associated with
said disease state and also dissolution of tau aggregates in the
brain of the patient or subject. As described in the Examples
below, the present inventors have shown that dissolution of such
aggregates is key effect in opening a clearance pathway (see e.g.
FIGS. 6A and 6B).
[0058] In one embodiment the cognitive disorder may be mild
cognitive impairment (MCI) e.g. amnestic MCI. Prior filed U.S.
provisional application 60/945,006 (herein specifically
incorporated by reference) describes the use of DAPTZ compounds for
MCI. While there is still discussion in the literature as to the
nature of the MCI concept (see Gauthier et al., Lancet, 2006; 367:
1262-1270; Petersen R C et al. Neuropathological features of
amnestic mild cognitive impairment. Arch Neurol 2006; 63: 665-672)
MCI is recognised as a valid disease target by the FDA. It is
defined by having a minor degree of cognitive impairment not yet
meeting clinical criteria for a diagnosis of dementia.
[0059] In one embodiment the CNS disorder may be a synucleinopathy
such as Parkinson's Disease (PD).
[0060] Prior filed PCT application PCT/GB2007/001105 (herein
specifically incorporated by reference) describes the use of DAPTZ
compounds for the treatment of PD and other synucleinopathies.
[0061] The synucleinopathies currently consist of the following
disorders: PD, dementia with Lewy bodies (DLB), multiple system
atrophy (MSA), drug-induced parkinsonism (e.g. produced by
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP] or pesticides
such as rotenone), and pure autonomic failure (PAF).
Patient Groups
[0062] Suitable subjects for the method may be selected on the
basis of conventional factors.
[0063] Thus, for example, for AD the initial selection of a patient
may involve any one or more of: rigorous evaluation by experienced
clinician; exclusion of non-AD diagnosis as far as possible by
supplementary laboratory and other investigations; objective
evaluation of level of cognitive function using neuropathologically
validated battery.
[0064] For MCI, representative criteria for syndromal MCI include
features: A. The patient is neither normal nor demented; B. There
is evidence of cognitive deterioration shown by either objectively
measured decline over time and/or subjective report of decline by
self and/or informant in conjunction with objective cognitive tests
(e.g. secondary tests if memory); C. Activities of daily living are
preserved and complex instrumental functions are either intact or
minimally impaired (see also Winblad, B. et al. (2004) Mild
cognitive impairment--beyond controversies, towards a concensus:
report of the International Working Group on Mild Cognitive
Impairment. J. Intern. Med. 256: 240-246). The patient will
generally be one diagnosed with MC1, but be one not diagnosed with
AD (i.e. will not show dementia). The patient may, for example, be
aged over 45, 50, 55 years. The patient may be one meeting one or
all of the following criteria in respect of: (i) Braak stage 3 or
less, 2 or less, 1 or less; (ii) MMSE score less than or equal to
MMSE 24, 25, 26, 27, 28 or 29, more preferably less than or equal
to MMSE 24, 25, 26, most preferably less than or equal to MMSE 24
or 25.
[0065] Diagnosis of PD is well known to those skilled in the
art.
[0066] As noted above, the methods of the present invention are
intended to treat a cognitive or CNS disorder in a patient such as
to maximise the relative cognitive or CNS benefit vs.
haematological effects of the DAPTZ compound.
[0067] In various aspects of the invention the patient may be one
whom is believed to be at above average risk of a haematological
disorder, the effects of which may otherwise be exacerbated by the
DAPTZ compound. Thus (without limitation) the patient may be one
known or believed to be suffering from a haemoglobinopathy such as
Sickle-cell disease, Thalassemia, Methaemoglobinemia; an anemia
(e.g. a haemolytic anemia); a haematological malignancy (e.g.
lymphoma, myeloma, plasmacytoma or leukemia); a coagulopathy such
as hemophilia; and so on. Above average risk of such diseases may
be assessed using conventional criteria e.g. symptomatic, genetic,
age, lifestyle, ethnicity (for example Sickle-cell disease occurs
more commonly in people--or their descendants--from parts of the
world such as sub-Saharan Africa). A particular class of patient at
risk of a haematological disorder would be those aged over 70 years
old, who may be subject to age-related anemic conditions (e.g.
myeloid dysplasia).
Dosage, Formulations and Delivery Vehicles
[0068] Within the disclosure herein, the precise selected dosage
level will depend on a variety of factors including, but not
limited to, the activity of the particular DAPTZ compound, the
duration of the treatment, other drugs, compounds, and/or materials
used in combination, the severity of the condition, and the
species, sex, age, weight, condition, general health, and prior
medical history of the patient.
[0069] Drug or dosage units (e.g., a pharmaceutical tablet or
capsule) with the appropriate loading, dissolution, or
gastroretention properties described above can be provided by those
skilled in the art based on the disclosure herein using
conventional technologies, and those conventional technologies do
not per se form part of the present invention.
[0070] For example rapid dissolution drug units (for oxidised or
reduced DAPTZ compounds) or slow or delayed release, dissolution
units (for reduced DAPTZ compounds) can be provided and tested to
order from commercial sources e.g. Encap Drug Delivery (Units 4, 5
& 6, Oakbank Park Way, Livingston, West Lothian, EH53 0TH,
Scotland, UK); Eurand (Via Martin Luther King, 13 20060, Pessano
con Bornago, Milan) and so on.
[0071] Gastro-retained drug units are also widely known in the
patent literature (e.g. U.S. Pat. No. 6,207,197, U.S. Pat. No.
5,972,389) and general literature, and have been for many
years--see e.g. Davis, et al., "Transit of pharmaceutical dosage
forms through the small intestine", Gut, 27 (8):886-892 (1986);
Fara, "Physiological limitations: gastric emptying and transit of
dosage forms" in: Rate Control in Drug Therapy, L. F. Prescott, et
al., Eds., Churchill Livingstone, New York (1985); Davis, S. S.
(2005) Formulation strategies for absorption windows Drug Discovery
Today 10:249-257. This latter notes that the process of GI transit
in humans and its implications for drug delivery are now well
understood. Dosage forms administered to a fed stomach will have
delayed emptying. A multiparticulate system, such as one containing
microspheres or pellets, can become mixed with the food and, as a
consequence, will usually empty with the food over an extended
period of time. If the administered particles are large, they will
not be able to pass through the constricted pylorus with the
digested food, and will have to wait until the stomach is empty and
in the fasted state. In general, particles up to 10 mm in size can
be expected to empty from the fed stomach. Exactly when the
particles empty will also depend on their number and their relative
positions within the stomach. Hence, a dosage form larger than
15-20 mm and administered with food is expected to achieve
gastroretention. Such a dosage form will then have an opportunity
to empty after the food has left the stomach when the fasted state
occurs.
[0072] A single unit system (or a multiparticulate) can empty
rapidly from the fasted stomach. Exactly when it will empty will
also depend on the timing of the housekeeper wave in relation to
dosing. The open pylorus has a diameter of 15 mm in humans. An
object greater than this size will have difficulty in passing into
the small intestine in the fasted (or fed) state. Based on this
knowledge, various approaches have been devised for
gastroretention. These fall into two main classes: (i) small
particles that have bioadhesive properties (and also a propensity
to float on the stomach contents); and (ii) large swelling objects
that will be retained in the stomach because of their size. These
swelling systems might also have floating characteristics, usually
provided by the generation of carbon dioxide.
[0073] The drug delivery company Depomed have described
gastroretentive tablets `that swell in the stomach which treats the
tablet like undigested food, and won't let it pass into the small
intestine. The tablet is retained by the stomach for several hours,
where it can deliver its payload of drug as quickly or slowly as
desired` (http://www.depomedinc.com/products_pipeline.htm).
[0074] These systems are based on polyethylene oxide (PEO) in
combination with hydroxypropyl methylcellulose (HPMC) to produce a
sustained-release matrix tablet that can swell. According to the
company, candidate molecules include metformin, gabapentin
ciprofloxacin and furosemide. Recent press releases state that
Depomed has completed Phase III clinical trials with once-daily
metformin for the treatment of Type II diabetes and with once-daily
ciprofloxacin for the treatment of urinary tract infections, and
that new drug applications (NDA) for both products have been filed
with the FDA. The company is also conducting a Phase II trial with
the diuretic furosemide.
[0075] A recent abstract has described a dual-labelled
scintigraphic study of controlled release furosemide gastric
retentive tablets in healthy volunteers. The dual-labelling
procedure permitted separate characterization of the erosion and
swelling. The tablets (and an immediate release control) were
administered after a high-fat breakfast. Gastric residence of the
swelling tablets was sufficiently long to deliver the drug to the
upper GI tract. Consequently, the plasma concentration of the drug
was extended and, furthermore, unlike previous slow release
formulations reported in the literature, there was no reduction in
bioavailability. From a standpoint of patient compliance, the
gastroretentive tablet provided gradual diuresis and natriuresis,
rather than the brief and intense diuresis of short onset time
experienced by patients taking conventional immediate release
furosemide tablets.
[0076] Thus known gastroretained dosage forms may be applicable to
the present invention, and in particular for use with oxidised
DAPTZ forms.
[0077] While it is possible for the diaminophenothiazinium compound
to be used (e.g., administered) alone, it is often preferable to
present it as a composition or formulation.
[0078] Preferably the drug or dosage unit is provided as a
pharmaceutical composition (e.g., formulation, preparation,
medicament) comprising the DAPTZ compound, as described herein, and
a pharmaceutically acceptable carrier, diluent, or excipient.
[0079] In one embodiment, the composition is a pharmaceutical
composition comprising at least one diaminophenothiazinium
compound, as described herein, together with one or more other
pharmaceutically acceptable ingredients well known to those skilled
in the art, including, but not limited to, pharmaceutically
acceptable carriers, diluents, excipients, adjuvants, fillers,
buffers, preservatives, anti-oxidants, lubricants, stabilisers,
solubilisers, surfactants (e.g., wetting agents), masking agents,
colouring agents, flavouring agents, and sweetening agents.
[0080] In one embodiment, the composition further comprises other
active agents, for example, other therapeutic or prophylactic
agents.
[0081] Suitable carriers, diluents, excipients, etc. can be found
in standard pharmaceutical texts. See, for example, Handbook of
Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash),
2001 (Synapse Information Resources, Inc., Endicott, New York,
USA), Remington's Pharmaceutical Sciences, 20th edition, pub.
Lippincott, Williams & Wilkins, 2000; and Handbook of
Pharmaceutical Excipients, 2nd edition, 1994.
[0082] The term "pharmaceutically acceptable," as used herein,
pertains to compounds, ingredients, materials, compositions, dosage
forms, etc., which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of the subject in
question (e.g., human) without excessive toxicity, irritation,
allergic response, or other problem or complication, commensurate
with a reasonable benefit/risk ratio. Each carrier, diluent,
excipient, etc. must also be "acceptable" in the sense of being
compatible with the other ingredients of the formulation.
[0083] The formulations may be prepared by any methods well known
in the art of pharmacy. Such methods include the step of bringing
into association the active compound with a carrier which
constitutes one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association the active compound with carriers (e.g., liquid
carriers, finely divided solid carrier, etc.), and then shaping the
product, if necessary.
[0084] As noted above, the formulation may be prepared to provide
for rapid or slow release; immediate, delayed, timed, or sustained
release; or a combination thereof.
Combination Therapies
[0085] Combination treatments and therapies, in which two or more
treatments or therapies are combined, for example, sequentially or
simultaneously, are discussed in more detail hereinafter. Thus it
will be understood that any of the medical uses or methods
described herein may be used in a combination therapy e.g. another
treatment for AD, MC1, or PD respectively. For example a treatment
of the invention for AD (e.g., employing a compound of the
invention) is in combination with a cholinesterase inhibitor such
as Donepezil (Aricept.TM.), Rivastigmine (Exelon.TM.) or
Galantamine (Reminyl.TM.).
[0086] In one embodiment, a treatment of the invention (e.g.,
employing a compound of the invention) is in combination with an
NMDA receptor antagonist such as Memantine (Ebixa.TM.,
Namenda.TM.).
[0087] In one embodiment, a treatment of the invention (e.g.
employing a compound of the invention) is in combination with a
muscarinic receptor agonist.
[0088] In one embodiment, a treatment of the invention (e.g.
employing a compound of the invention) is in combination with an
inhibitor of amyloid precursor protein to beta-amyloid (e.g., an
inhibitor of amyloid precursor protein processing that leads to
enhanced generation of beta-amyloid).
Example DAPTZ Compounds
[0089] The relationship between oxidised and reduced DAPTZ
compounds can be conveniently illustrated using MTC, a
phenothiazin-5-ium salt. This may conveniently be considered to be
an "oxidized form" when considered in respect of the corresponding
10H-phenothiazine compound,
N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diamine, which may
conveniently be considered to be a "reduced form":
##STR00002##
[0090] This aspect of the invention pertains to certain
diaminophenothiazine compounds and analogs thereof, having one of
the following formulae, and pharmaceutically acceptable salts,
hydrates, and solvates thereof (collectively referred to herein as
"diaminophenothiazines" or "diaminophenothiazine compounds"):
##STR00003##
[0091] Formula (1) depicts compounds in a reduced form, whereas
each of Formulae (2), (3), and (4) depicts compounds in an oxidized
form.
[0092] In one embodiment, the compounds are selected from compounds
of formula (1), and pharmaceutically acceptable salts, hydrates,
and solvates thereof.
[0093] In one embodiment, the compounds are selected from compounds
of formula (2) or (3), and pharmaceutically acceptable salts,
hydrates, and solvates thereof.
[0094] In one embodiment, the compounds are selected from compounds
of formula (4), and pharmaceutically acceptable salts, hydrates,
and solvates thereof.
[0095] Each one of the above structures is only one of many
equivalent resonance structures, and all of which are intended to
be encompassed by that representative structure. For example,
structure (4) is only one of many equivalent resonance structures,
some of which are shown below, and all of which are intended to be
encompassed by structure (4):
##STR00004##
Carbon Ring Atom Substituents
[0096] In each one of the above formulae, each one of R.sup.1,
R.sup.2, R.sup.4, R.sup.6, R.sup.8, and R.sup.9 is independently
selected from: [0097] --H; [0098] --F; --Cl; --Br; --I; [0099]
--OH; --OR; [0100] --SH; --SR; [0101] --NO.sub.2; [0102]
--C(.dbd.O)R; [0103] --C(.dbd.O)OH; --C(.dbd.O)OR; [0104]
--C(.dbd.O)NH.sub.2; --C(.dbd.O)NHR; --C(.dbd.O)NR.sub.2;
--C(.dbd.O)NR.sup.N1R.sup.N2; [0105] --NH.sub.2; --NHR; --NR.sub.2;
--NR.sup.N1R.sup.N2; [0106] --NHC(.dbd.O)H; --NRC(.dbd.O)H;
--NHC(.dbd.O)R; --NRC(.dbd.O)R; [0107] --R; wherein each R is
independently selected from: [0108] unsubstituted aliphatic
C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl; [0109]
unsubstituted aliphatic C.sub.2-6alkenyl; substituted aliphatic
C.sub.2-6alkenyl; [0110] unsubstituted C.sub.3-6cycloalkyl;
substituted C.sub.3-6cycloalkyl; [0111] unsubstituted
C.sub.6-10-carboaryl; substituted C.sub.6-10-carboaryl; [0112]
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; [0113] unsubstituted
C.sub.6-10-carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10-carboaryl-C.sub.1-4alkyl; wherein, in each group
--NR.sup.N1R.sup.N2, independently, R.sup.N1 and R.sup.N2 taken
together with the nitrogen atom to which they are attached form a
ring having from 3 to 7 ring atoms.
[0114] Examples of groups --NR.sup.N1R.sup.N2, wherein R.sup.N1 and
R.sup.N2 taken together with the nitrogen atom to which they are
attached form a ring having from 3 to 7 ring atoms, include:
pyrrolidino, piperidino, piperazino, morpholino, pyrrolyl, and
substituted forms, such as N-substituted forms, such as N-methyl
piperazino.
[0115] In one embodiment, each one of R.sup.1, R.sup.2, R.sup.4,
R.sup.6, R.sup.8, and R.sup.9 is independently selected from:
[0116] --H; [0117] --F; --Cl; --Br; --I; [0118] --OH; --OR; [0119]
--C(.dbd.O)OH; --C(.dbd.O)OR; [0120] --R.
[0121] In one embodiment, each one of R.sup.1, R.sup.2, R.sup.4,
R.sup.6, R.sup.8, and R.sup.9 is independently selected from:
[0122] --H; [0123] --R.
[0124] In one embodiment, each R is independently selected from:
[0125] unsubstituted aliphatic C.sub.1-6alkyl; substituted
aliphatic C.sub.1-6alkyl; [0126] unsubstituted aliphatic
C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl; [0127]
unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl.
[0128] In one embodiment, each R is independently selected from:
[0129] unsubstituted aliphatic C.sub.1-6alkyl; substituted
aliphatic C.sub.1-6alkyl.
[0130] In one embodiment, each R is independently selected from:
-Me, -Et, -nPr, and -iPr.
[0131] In one embodiment, each R is independently selected from:
-Me and -Et.
[0132] In one embodiment, the C.sub.1-6alkyl group is a
C.sub.1-4alkyl group.
[0133] In one embodiment, the C.sub.2-6alkenyl group is a
C.sub.2-4alkenyl group.
[0134] In one embodiment, the C.sub.3-6cycloalkyl group is a
C.sub.3-4cycloalkyl group.
[0135] Examples of unsubstituted aliphatic C.sub.1-6alkyl groups
include: methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,
sec-butyl, tert-butyl, n-pentyl, iso-pentyl, tert-pentyl,
neo-pentyl, hexyl, iso-hexyl, etc.
[0136] Examples of unsubstituted aliphatic C.sub.2-6alkenyl groups
include: propen-1-yl, propen-2-yl, buten-1-yl, buten-2-yl,
buten-3-yl, etc.
[0137] Examples of unsubstituted C.sub.3-6cycloalkyl groups
include: cyclopropyl, cyclopropyl-methyl, cyclobutyl, cyclopentyl,
cyclohexyl, etc.
[0138] In one embodiment, the C.sub.6-10-carboaryl group is a
C.sub.6carboaryl group.
[0139] In one embodiment, the C.sub.5-10heteroaryl group is a
C.sub.5-6heteroaryl group.
[0140] In one embodiment, the C.sub.6-10-carboaryl-C.sub.1-4alkyl
group is a C.sub.6carboaryl-C.sub.1-2alkyl group.
[0141] Examples of unsubstituted C.sub.6-10carboaryl groups
include: phenyl, naphthyl.
[0142] Examples of unsubstituted C.sub.5-10heteroaryl groups
include: pyrrolyl, thienyl, furyl, imidazolyl, oxazolyl,
isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, pyridyl, pyrazinyl,
pyrimidinyl, pyridazinyl.
[0143] Examples of unsubstituted C.sub.6-10carboaryl-C.sub.1-4alkyl
groups include: benzyl, phenylethyl.
[0144] In one embodiment, optional substituents (e.g., on aliphatic
C.sub.1-6alkyl, aliphatic C.sub.1-6alkenyl, C.sub.3-6cycloalkyl,
C.sub.6-10carboaryl, C.sub.5-10heteroaryl,
C.sub.6-10carboaryl-C.sub.1-4alkyl) are independently selected
from: [0145] --F; --Cl; --Br; --I; [0146] --OH; --OR'; [0147] --SH;
--SR'; [0148] --NO.sub.2; [0149] --C(.dbd.O)R'; [0150]
--C(.dbd.O)OH; --C(.dbd.O)OR'; [0151] --C(.dbd.O)NH.sub.2;
--C(.dbd.O)NHR'; --C(.dbd.O)NR'.sub.2;
--C(.dbd.O)NR'.sup.N1R'.sup.N2; [0152] --NH.sub.2; --NHR';
--NR'.sub.2; --NR'.sup.N1R'.sup.N2; [0153] --NHC(.dbd.O)H;
--N'RC(.dbd.O)H; --NHC(.dbd.O)'R; --N'RC(.dbd.O)'R; [0154] --R';
wherein each R' is independently selected from: [0155]
unsubstituted aliphatic C.sub.1-6alkyl; substituted aliphatic
C.sub.1-6alkyl; [0156] unsubstituted aliphatic C.sub.2-6alkenyl;
substituted aliphatic C.sub.2-6alkenyl; [0157] unsubstituted
C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl; [0158]
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
[0159] unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; [0160] unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; wherein, in each group
--NR'.sup.N1R'.sup.N2, independently, R'.sup.N1 and R'.sup.N2 taken
together with the nitrogen atom to which they are attached form a
ring having from 3 to 7 ring atoms.
[0161] In one embodiment, optional substituents (e.g., on aliphatic
C.sub.1-6alkyl, aliphatic C.sub.1-6alkenyl, C.sub.3-6cycloalkyl,
C.sub.6-10carboaryl, C.sub.6-10heteroaryl,
C.sub.6-10carboaryl-C.sub.1-4alkyl) are independently selected
from: [0162] --F; --Cl; --Br; --I; [0163] --OH; --OR; [0164]
--C(.dbd.O)OH; --C(.dbd.O)OR'; [0165] --R'.
[0166] In one embodiment, optional substituents (e.g., on aliphatic
C.sub.1-6alkyl, aliphatic C.sub.1-6alkenyl, C.sub.3-6cycloalkyl,
C.sub.6-10carboaryl, C.sub.5-10heteroaryl,
C.sub.6-10carboaryl-C.sub.1-4alkyl) are as defined above, except
that each R' is independently selected from: [0167] unsubstituted
aliphatic C.sub.1-6alkyl; [0168] unsubstituted aliphatic
C.sub.2-6alkenyl; [0169] unsubstituted C.sub.3-6cycloalkyl; [0170]
unsubstituted C.sub.6-10-carboaryl; [0171] unsubstituted
C.sub.5-10heteroaryl; [0172] unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl.
[0173] In one embodiment, optional substituents (e.g., on aliphatic
C.sub.1-6alkyl, aliphatic C.sub.1-6alkenyl, C.sub.3-6cycloalkyl,
C.sub.6-10carboaryl, C.sub.5-10heteroaryl,
C.sub.6-10carboaryl-C.sub.1-4alkyl) are as defined above, except
that each R' is independently selected from: [0174] unsubstituted
aliphatic C.sub.1-6alkyl; [0175] unsubstituted aliphatic
C.sub.2-6alkenyl; [0176] unsubstituted C.sub.3-6cycloalkyl.
[0177] In one embodiment, optional substituents (e.g., on aliphatic
C.sub.1-6alkyl, aliphatic C.sub.1-6alkenyl, C.sub.3-6cycloalkyl,
C.sub.6-10carboaryl, C.sub.5-10heteroaryl,
C.sub.6-10carboaryl-C.sub.1-4alkyl) are as defined above, except
that each R' is independently selected from: [0178] unsubstituted
aliphatic C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl.
[0179] In one embodiment, optional substituents (e.g., on aliphatic
C.sub.1-6alkyl, aliphatic C.sub.1-6alkenyl, C.sub.3-6cycloalkyl,
C.sub.6-10carboaryl, C.sub.5-10heteroaryl,
C.sub.6-10carboaryl-C.sub.1-4alkyl) are as defined above, except
that each R' is independently selected from: -Me, -Et, -nPr, and
-iPr.
[0180] In one embodiment, optional substituents (e.g., on aliphatic
C.sub.1-6alkyl, aliphatic C.sub.1-6alkenyl, C.sub.3-6cycloalkyl,
C.sub.6-10carboaryl, C.sub.5-10heteroaryl,
C.sub.6-10carboaryl-C.sub.1-4alkyl) are as defined above, except
that each R' is independently selected from: -Me and -Et.
[0181] In one embodiment, each one of R.sup.1, R.sup.2, R.sup.4,
R.sup.6, R.sup.8, and R.sup.9 is independently selected from: --H,
-Me, -Et, -nPr, and -iPr.
[0182] In one embodiment, each one of R.sup.1, R.sup.2, R.sup.4,
R.sup.6, R.sup.8, and R.sup.9 is independently selected from: --H,
-Me, and -Et.
[0183] In one embodiment, each one of R.sup.1, R.sup.2, R.sup.4,
R.sup.6, R.sup.8, and R.sup.9 is independently selected from: --H
and -Me.
[0184] In one embodiment, all except two of R.sup.1, R.sup.2,
R.sup.4, R.sup.6, R.sup.8, and R.sup.9 is --H.
[0185] In one embodiment, all except one of R.sup.1, R.sup.2,
R.sup.4, R.sup.6, R.sup.8, and R.sup.9 is --H.
[0186] In one embodiment, each of R.sup.1, R.sup.2, R.sup.4,
R.sup.6, R.sup.8, and R.sup.9 is --H.
Amino Groups
[0187] In each one of the above formulae, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently --H or as defined above for R; or
R.sup.3NA and R.sup.3NB taken together with the nitrogen atom to
which they are attached form a ring having from 3 to 7 ring
atoms.
[0188] For example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently as defined above for R; or R.sup.3NA and
R.sup.3NB taken together with the nitrogen atom to which they are
attached form a ring having from 3 to 7 ring atoms.
[0189] For example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: [0190] --H; [0191]
unsubstituted aliphatic C.sub.1-6alkyl; substituted aliphatic
C.sub.1-6alkyl; [0192] unsubstituted aliphatic C.sub.2-6alkenyl;
substituted aliphatic C.sub.2-6alkenyl; [0193] unsubstituted
C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl; [0194]
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; or R.sup.3NA and R.sup.3NB
taken together with the nitrogen atom to which they are attached
form a ring having from 3 to 7 ring atoms.
[0195] For example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: [0196] unsubstituted
aliphatic C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl;
[0197] unsubstituted aliphatic C.sub.2-6alkenyl; substituted
aliphatic C.sub.2-6alkenyl; [0198] unsubstituted
C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl; [0199]
unsubstituted C.sub.6-10carboaryl; substituted C.sub.6-10carboaryl;
[0200] unsubstituted C.sub.5-10heteroaryl; substituted
C.sub.5-10heteroaryl; [0201] unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; or R.sup.3NA and R.sup.3NB
taken together with the nitrogen atom to which they are attached
form a ring having from 3 to 7 ring atoms.
[0202] In another example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: [0203] --H; [0204]
unsubstituted aliphatic C.sub.1-6alkyl; substituted aliphatic
C.sub.1-6alkyl; [0205] unsubstituted aliphatic C.sub.2-6alkenyl;
substituted aliphatic C.sub.2-6alkenyl; [0206] unsubstituted
C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl; or R.sup.3NA
and R.sup.3NB taken together with the nitrogen atom to which they
are attached form a ring having from 3 to 7 ring atoms.
[0207] In another example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: [0208] unsubstituted
aliphatic C.sub.1-6alkyl; substituted aliphatic C.sub.1-6alkyl;
[0209] unsubstituted aliphatic C.sub.2-6alkenyl; substituted
aliphatic C.sub.2-6alkenyl; [0210] unsubstituted
C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl; or R.sup.3NA
and R.sup.3NB taken together with the nitrogen atom to which they
are attached form a ring having from 3 to 7 ring atoms.
[0211] In another example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: [0212] --H; [0213]
unsubstituted aliphatic C.sub.1-6alkyl; [0214] unsubstituted
aliphatic C.sub.2-6alkenyl; [0215] unsubstituted
C.sub.3-6cycloalkyl; or R.sup.3NA and R.sup.3NB taken together with
the nitrogen atom to which they are attached form a ring having
from 3 to 7 ring atoms.
[0216] In another example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: [0217] unsubstituted
aliphatic C.sub.7-6alkyl; [0218] unsubstituted aliphatic
C.sub.2-6alkenyl; [0219] unsubstituted C.sub.3-6cycloalkyl; or
R.sup.3NA and R.sup.3NB taken together with the nitrogen atom to
which they are attached form a ring having from 3 to 7 ring
atoms.
[0220] In another example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: --H, -Me, -Et, -nPr, and
-iPr.
[0221] In another example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: --H, -Me, and -Et (e.g.,
--NR.sup.3NAR.sup.3NA is --NH.sub.2, --NHMe, --NMe.sub.2, --NHEt,
--NEt.sub.2, or --NMeEt).
[0222] In another example, in one embodiment, in each group
--NR.sup.3NAR.sup.3NB, if present, each one of R.sup.3NA and
R.sup.3NB is independently selected from: --H and -Me (e.g.,
--NR.sup.3NAR.sup.3NA is --NH.sub.2, --NHMe, or --NMe.sub.2).
[0223] In precise analogy, in each one of the above formulae, in
each group --NR.sup.7NAR.sup.7NB, if present, each one of R.sup.7NA
and R.sup.7NB is independently --H or as defined above for R; or
R.sup.7NA and R.sup.7NB taken together with the nitrogen atom to
which they are attached form a ring having from 3 to 7 ring
atoms.
[0224] For example, in one embodiment, in each group
--NR.sup.7NAR.sup.7NB, if present, each one of R.sup.7NA and
R.sup.7NB is independently as defined above for R; or R.sup.7NA and
R.sup.7NB taken together with the nitrogen atom to which they are
attached form a ring having from 3 to 7 ring atoms.
[0225] In one embodiment, --NR.sup.3NAR.sup.3NB and
--NR.sup.7NAR.sup.7NB, if both present, are the same.
[0226] In one embodiment, --NR.sup.3NAR.sup.3NB and
--NR.sup.7NAR.sup.7NB, if both present, are different.
[0227] In each one of the above formulae, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently --H or as
defined above for R.
[0228] For example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently as defined
above for R.
[0229] For example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: [0230] --H; [0231] unsubstituted aliphatic C.sub.1-6alkyl;
substituted aliphatic C.sub.1-6alkyl; [0232] unsubstituted
aliphatic C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
[0233] unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl; [0234] unsubstituted C.sub.6-10carboaryl;
substituted C.sub.6-10carboaryl; [0235] unsubstituted
C.sub.5-10heteroaryl; substituted C.sub.5-10heteroaryl; [0236]
unsubstituted C.sub.6-10-carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl.
[0237] For example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: [0238] unsubstituted aliphatic C.sub.1-6alkyl; substituted
aliphatic C.sub.1-6alkyl; [0239] unsubstituted aliphatic
C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl; [0240]
unsubstituted C.sub.3-6cycloalkyl; substituted C.sub.3-6cycloalkyl;
[0241] unsubstituted C.sub.6-10carboaryl; substituted
C.sub.6-10carboaryl; [0242] unsubstituted C.sub.5-10heteroaryl;
substituted C.sub.5-10heteroaryl; [0243] unsubstituted
C.sub.6-10carboaryl-C.sub.1-4alkyl; substituted
C.sub.6-10carboaryl-C.sub.1-4alkyl.
[0244] In another example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: [0245] --H; [0246] unsubstituted aliphatic C.sub.1-6alkyl;
substituted aliphatic C.sub.1-6alkyl; [0247] unsubstituted
aliphatic C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl;
[0248] unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl.
[0249] In another example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: [0250] unsubstituted aliphatic C.sub.1-6alkyl; substituted
aliphatic C.sub.1-6alkyl; [0251] unsubstituted aliphatic
C.sub.2-6alkenyl; substituted aliphatic C.sub.2-6alkenyl; [0252]
unsubstituted C.sub.3-6cycloalkyl; substituted
C.sub.3-6cycloalkyl.
[0253] In another example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: [0254] --H; [0255] unsubstituted aliphatic C.sub.1-6alkyl;
[0256] unsubstituted aliphatic C.sub.2-6alkenyl; [0257]
unsubstituted C.sub.3-6cycloalkyl.
[0258] In another example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: [0259] unsubstituted aliphatic C.sub.1-6alkyl; [0260]
unsubstituted aliphatic C.sub.2-6alkenyl; [0261] unsubstituted
C.sub.3-6cycloalkyl.
[0262] In another example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: --H, -Me, -Et, -nPr, and -iPr.
[0263] In another example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: --H, -Me, and -Et (e.g., .dbd.NR.sup.3NC is .dbd.NH,
.dbd.NMe, or .dbd.NEt).
[0264] In another example, in one embodiment, in each group
.dbd.NR.sup.3NC, if present, R.sup.3NC is independently selected
from: --H and -Me (e.g., .dbd.NR.sup.3NC is .dbd.NH or
.dbd.NMe).
[0265] In precise analogy, in each one of the above formulae, in
each group .dbd.NR.sup.7NC, if present, e.sup.7NC is independently
as defined above for R.sup.3NC.
Nitrogen Ring Atom Substituent
[0266] Also, in precise analogy, in each one of the above formulae,
R.sup.N10, if present, is independently as defined above for
R.sup.3NC (or R.sup.7NC).
[0267] For example, in one embodiment, R.sup.N10, if present, is
independently selected from: --H and unsubstituted aliphatic
C.sub.1-6alkyl.
[0268] For example, in one embodiment, R.sup.N10, if present, is
independently selected from: --H, -Me, and -Et.
[0269] For example, in one embodiment, R.sup.N10, if present, is
independently selected from: --H and -Me.
[0270] For example, in one embodiment, R.sup.N10, if present, is
independently --H.
Counter Ion
[0271] X.sup.-, if present, is one or more anionic counter ions to
achieve electrical neutrality.
[0272] Examples of suitable anionic counter ions are discussed
below under the heading "Salts".
[0273] In one embodiment, X.sup.- is independently a halogen anion
(i.e., a halide).
[0274] In one embodiment, X.sup.- is independently Cl.sup.-,
Br.sup.-, or I.sup.-.
[0275] In one embodiment, X.sup.- is independently Cl.sup.-.
[0276] In one embodiment, X.sup.- is independently
NO.sub.3.sup.-.
Isomers
[0277] Certain compounds may exist in one or more particular
geometric, optical, enantiomeric, diasteriomeric, epimeric,
atropic, stereoisomeric, tautomeric, conformational, or anomeric
forms, including but not limited to, cis- and trans-forms; E- and
Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and
meso-forms; D- and L-forms; d- and I-forms; (+) and (-) forms;
keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal-
and anticlinal-forms; .alpha.- and .beta.-forms; axial and
equatorial forms; boat-, chair-, twist-, envelope-, and
halfchair-forms; and combinations thereof, hereinafter collectively
referred to as "isomers" (or "isomeric forms").
[0278] Note that, except as discussed below for tautomeric forms,
specifically excluded from the term "isomers," as used herein, are
structural (or constitutional) isomers (i.e., isomers which differ
in the connections between atoms rather than merely by the position
of atoms in space). For example, a reference to a methoxy group,
--OCH.sub.3, is not to be construed as a reference to its
structural isomer, a hydroxymethyl group, --CH.sub.2OH. Similarly,
a reference to ortho-chlorophenyl is not to be construed as a
reference to its structural isomer, meta-chlorophenyl. However, a
reference to a class of structures may well include structurally
isomeric forms falling within that class (e.g., C.sub.1-7alkyl
includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-,
and tert-butyl; methoxyphenyl includes ortho-, meta-, and
para-methoxyphenyl).
[0279] The above exclusion does not pertain to tautomeric forms,
for example, keto-, enol-, and enolate-forms, as in, for example,
the following tautomeric pairs: keto/enol (illustrated below),
imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime,
thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.
##STR00005##
[0280] Note that specifically included in the term "isomer" are
compounds with one or more isotopic substitutions. For example, H
may be in any isotopic form, including .sup.1H, .sup.2H (D), and
.sup.3H (T); C may be in any isotopic form, including .sup.11C,
.sup.12C, .sup.13C, and .sup.14C; O may be in any isotopic form,
including .sup.16O and .sup.18O; and the like.
[0281] Unless otherwise specified, a reference to a particular
compound includes all such isomeric forms, including (wholly or
partially) racemic and other mixtures thereof. Methods for the
preparation (e.g., asymmetric synthesis) and separation (e.g.,
fractional crystallisation and chromatographic means) of such
isomeric forms are either known in the art or are readily obtained
by adapting the methods taught herein, or known methods, in a known
manner.
Salts
[0282] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding salt of the compound, for example, a
pharmaceutically-acceptable salt. Examples of pharmaceutically
acceptable salts are discussed in Berge et al., 1977,
"Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp.
1-19.
[0283] For example, if the compound is anionic, or has a functional
group which may be anionic (e.g., --COON may be --COO.sup.-), then
a salt may be formed with a suitable cation. Examples of suitable
inorganic cations include, but are not limited to, alkali metal
ions such as Na.sup.+ and K.sup.+, alkaline earth cations such as
Ca.sup.2+ and Mg.sup.2+, and other cations such as Al.sup.3+.
Examples of suitable organic cations include, but are not limited
to, ammonium ion (i.e., NH.sub.4.sup.+) and substituted ammonium
ions (e.g., NH.sub.3, R.sup.+, NH.sub.2R.sub.2.sup.+,
NHR.sub.3.sup.+, NR.sub.4.sup.+). Examples of some suitable
substituted ammonium ions are those derived from: ethylamine,
diethylamine, dicyclohexylamine, triethylamine, butylamine,
ethylenediamine, ethanolamine, diethanolamine, piperazine,
benzylamine, phenylbenzylamine, choline, meglumine, and
tromethamine, as well as amino acids, such as lysine and arginine.
An example of a common quaternary ammonium ion is
N(CH.sub.3).sub.4.sup.+.
[0284] If the compound is cationic, or has a functional group which
may be cationic (e.g., --NH.sub.2 may be --NH.sub.3.sup.+), then a
salt may be formed with a suitable anion. Examples of suitable
inorganic anions include, but are not limited to, those derived
from the following inorganic acids: hydrochloric, hydrobromic,
hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and
phosphorous.
[0285] Examples of suitable organic anions include, but are not
limited to, those derived from the following organic acids:
2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic,
camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic,
ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic,
glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic,
lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic,
oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic,
phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic,
sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of
suitable polymeric organic anions include, but are not limited to,
those derived from the following polymeric acids: tannic acid,
carboxymethyl cellulose.
[0286] The compound may also be provided in the form of a mixed
salt (i.e., the compound in combination with a salt, or another
salt). For example, methyl-thioninium chloride zinc chloride mixed
salt (MTZ) is a mixed salt of methyl-thioninium chloride (MTC), a
chloride salt, and another salt, zinc chloride. Such mixed salts
are intended to be encompassed by the term "and pharmaceutically
acceptable salts thereof".
[0287] Unless otherwise specified, a reference to a particular
compound also includes salt forms thereof.
Hydrates and Solvates
[0288] It may be convenient or desirable to prepare, purify, and/or
handle a corresponding solvate of the active compound. The term
"solvate" is used herein in the conventional sense to refer to a
complex of solute (e.g., compound, salt of compound) and solvent.
If the solvent is water, the solvate may be conveniently referred
to as a hydrate, for example, a mono-hydrate, a di-hydrate, a
tri-hydrate, etc.
[0289] Unless otherwise specified, a reference to a particular
compound also includes solvate forms thereof.
[0290] In all embodiments, a preferred oxidised
diaminophenothiazine is MTC.
Preferred Stable Crystalline Reduced DAPTZ Compounds
[0291] Preferred compounds are described in prior filed,
unpublished, application PCT/GB2007/001103, and are
3,7-diamino-10H-phenothiazine compounds of the following
formula:
##STR00006##
[0292] wherein: [0293] each of R.sup.1 and R.sup.9 is independently
selected from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and
halogenated C.sub.1-4alkyl; [0294] each of R.sup.3NA and R.sup.3NB
is independently selected from: --H, C.sub.1-4alkyl,
C.sub.2-4alkenyl, and halogenated C.sub.1-4alkyl; [0295] each of
R.sup.7NA and R.sup.7NB is independently selected from: --H,
C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated C.sub.1-4alkyl;
[0296] each of HX.sup.1 and HX.sup.2 is independently a protic
acid; and pharmaceutically acceptable salts, solvates, and hydrates
thereof.
[0297] Without wishing to be bound to any particular theory, the
inventors believe that it is possible, if not likely, that the
compounds exist in the following form:
##STR00007##
[0298] Although the DAPTZ compounds are themselves salts, they may
also be provided in the form of a mixed salt (i.e., the DAPTZ in
combination with another salt). Such mixed salts are intended to be
encompassed by the term "and pharmaceutically acceptable salts
thereof". Unless otherwise specified, a reference to a particular
compound also includes salts thereof.
[0299] The DAPTZ compounds may also be provided in the form of a
solvate or hydrate. The term "solvate" is used herein in the
conventional sense to refer to a complex of solute (e.g., compound,
salt of compound) and solvent. If the solvent is water, the solvate
may be conveniently referred to as a hydrate, for example, a
mono-hydrate, a di-hydrate, a tri-hydrate, etc. Unless otherwise
specified, a reference to a particular compound also includes
solvate forms thereof.
[0300] In one embodiment, the C.sub.1-4alkyl groups are selected
from: linear C.sub.1-4alkyl groups, such as -Me, -Et, -nPr, -iPr,
and -nBu; branched C.sub.3-4alkyl groups, such as -iPr, -iBu, -sBu,
and -tBu; and cyclic C.sub.3-4alkyl groups, such as -cPr and
-cBu.
[0301] In one embodiment, the C.sub.2-4alkenyl groups are selected
from linear C.sub.1-4alkenyl groups, such as --CH.dbd.CH.sub.2
(vinyl) and --CH.sub.2--CH.dbd.CH.sub.2 (allyl).
[0302] In one embodiment, the halogenated C.sub.1-4alkyl groups are
selected from: --CF.sub.3, --CH.sub.2CF.sub.3, and
--CF.sub.2CF.sub.3.
The Groups R.sup.1 and R.sup.9
[0303] In one embodiment, each of R.sup.1 and R.sup.9 is
independently --H, -Me, -Et, or --CF.sub.3.
[0304] In one embodiment, each of R.sup.1 and R.sup.9 is
independently --H, -Me, or -Et.
[0305] In one embodiment, R.sup.1 and R.sup.9 are the same.
[0306] In one embodiment, R.sup.1 and R.sup.9 are different.
[0307] In one embodiment, each of R.sup.1 and R.sup.9 is
independently --H.
[0308] In one embodiment, each of R.sup.1 and R.sup.9 is
independently -Me.
[0309] In one embodiment, each of R.sup.1 and R.sup.9 is
independently -Et.
The Groups R.sup.3NA and R.sup.3NB
[0310] Each of R.sup.3NA and R.sup.3NB is independently selected
from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl.
[0311] In one embodiment, each of R.sup.3NA and R.sup.3NB is
independently selected from: C.sub.1-4alkyl, C.sub.2-4alkenyl, and
halogenated C.sub.1-4alkyl.
[0312] In one embodiment, each of R.sup.3NA and R.sup.3NB is
independently -Me, -Et, -nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or
--CF.sub.3.
[0313] In one embodiment, each of R.sup.3NA and R.sup.3NB is
independently -Me, -nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or
--CF.sub.3.
[0314] In one embodiment, each of R.sup.3NA and R.sup.3NB is
independently -Me or -Et.
[0315] In one embodiment, R.sup.3NA and R.sup.3NB are the same.
[0316] In one embodiment, R.sup.3NA and R.sup.3NB are
different.
[0317] In one embodiment, each of R.sup.3NA and R.sup.3NB is
independently -Me.
[0318] In one embodiment, each of R.sup.3NA and R.sup.3NB is
independently -Et.
[0319] The Groups R.sup.7NA and R.sup.7NB
[0320] Each of R.sup.7NA and R.sup.7NB is independently selected
from: --H, C.sub.1-4alkyl, C.sub.2-4alkenyl, and halogenated
C.sub.1-4alkyl.
[0321] In one embodiment, each of R.sup.7NA and R.sup.7NB is
independently selected from: C.sub.1-4alkyl, C.sub.2-4alkenyl, and
halogenated C.sub.1-4alkyl.
[0322] In one embodiment, each of R.sup.7NA and R.sup.7NB is
independently -Me, -Et, -nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or
--CF.sub.3.
[0323] In one embodiment, each of R.sup.7NA and R.sup.7NB is
independently -Me, -nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or
--CF.sub.3.
[0324] In one embodiment, each of R.sup.7NA and R.sup.7NB is
independently -Me or -Et.
[0325] In one embodiment, R.sup.7NA and R.sup.7NB are the same.
[0326] In one embodiment, R.sup.7NA and R.sup.7NB are
different.
[0327] In one embodiment, each of R.sup.7NA and R.sup.7NB is
independently -Me.
[0328] In one embodiment, each of R.sup.7NA and R.sup.7NB is
independently -Et.
[0329] In one embodiment, R.sup.3NA and R.sup.3NB and R.sup.7NA and
R.sup.7NB are the same.
[0330] In one embodiment, R.sup.3NA and R.sup.3NB and R.sup.7NA and
R.sup.MB are as defined herein, with the proviso that at least one
of R.sup.3NA and R.sup.3NB and R.sup.7NA and R.sup.7NB is other
than -Et.
Optional Provisos
[0331] In one embodiment, the compound is as defined herein, but
with the proviso that:
[0332] R.sup.3NA and R.sup.3NB and R.sup.7NA and R.sup.7NB are not
each -Et.
[0333] In one embodiment, the compound is as defined herein, but
with the proviso that:
[0334] if: each of R.sup.1 and R.sup.9 is --H;
[0335] then: R.sup.3NA and R.sup.3NB and R.sup.7NA and R.sup.7NB
are not each -Et.
The Groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB)
[0336] In one embodiment:
[0337] each of R.sup.3NA and R.sup.3NB is independently
C.sub.1-4alkyl, C.sub.2-4alkenyl, or halogenated
C.sub.1-4alkyl;
[0338] each of R.sup.7NA and R.sup.7NB is independently
C.sub.1-4alkyl, C.sub.2-4alkenyl, or halogenated
C.sub.1-4alkyl;
[0339] optionally with the proviso that at least one of R.sup.3NA
and R.sup.3NB and R.sup.7NA and R.sup.7NB is other than -Et.
[0340] In one embodiment:
[0341] each of R.sup.3NA and R.sup.3NB is independently -Me, -Et,
-nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or --CF.sub.3;
[0342] each of R.sup.7NA and R.sup.7NB is independently -Me, -Et,
-nPr, -nBu, --CH.sub.2--CH.dbd.CH.sub.2, or --CF.sub.3; optionally
with the proviso that at least one of R.sup.3NA and R.sup.3NB and
R.sup.7NA and R.sup.7NB is other than -Et.
[0343] In one embodiment:
[0344] each of R.sup.3NA and R.sup.3NB is independently -Me or
-Et;
[0345] each of R.sup.7NA and R.sup.7NB is independently -Me or
-Et;
[0346] optionally with the proviso that at least one of R.sup.3NA
and R.sup.3NB and R.sup.7NA and R.sup.7NB is other than -Et.
[0347] In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same.
[0348] In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are different.
[0349] In one embodiment, each of the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) is
independently selected from: --NMe.sub.2, --NEt.sub.2,
--N(nPr).sub.2, --N(Bu).sub.2, --NMeEt, --NMe(nPr), and
--N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
[0350] In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same, and are independently
selected from: --NMe.sub.2, --NEt.sub.2, --N(nPr).sub.2,
--N(Bu).sub.2, --NMeEt, --NMe(nPr), and
--N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
[0351] In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same, and are independently
selected from: --NMe.sub.2 and --NEt.sub.2.
[0352] In one embodiment, each of the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) is:
--NMe.sub.2.sup.+.
[0353] In one embodiment, at least one of the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) is other
than --NEt.sub.2.
[0354] In one embodiment, each of the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) is other
than --NEt.sub.2.
[0355] For example, in one embodiment, the groups
--N(R.sup.3NA)(R.sup.3NB) and --N(R.sup.7NA)(R.sup.7NB) are the
same, and are selected from: --NMe.sub.2, --N(nPr).sub.2,
--N(Bu).sub.2, --NMeEt, --NMe(nPr), and
--N(CH.sub.2CH.dbd.CH.sub.2).sub.2.
The Groups HX.sup.1 and HX.sup.2
[0356] Each of HX.sup.1 and HX.sup.2 is independently a protic
acid.
[0357] Examples of protic acids include, for example, inorganic
acids, such as hydrohalide acids (e.g., HCl, HBr, HI), nitric acid
(HNO.sub.3), sulphuric acid (H.sub.2SO.sub.4), and organic acids,
such as carbonic acid (H.sub.2CO.sub.3) and acetic acid
(CH.sub.3COOH).
[0358] In one embodiment, each of HX.sup.1 and HX.sup.2 is
independently a monoprotic acid.
[0359] In one embodiment, each of HX.sup.1 and HX.sup.2 is
independently a hydrohalide acid (i.e., a hydrohalic acid)
[0360] In one embodiment, each of HX.sup.1 and HX.sup.2 is
independently selected from HCl, HBr, and HI.
[0361] In one embodiment, HX.sup.1 and HX.sup.2 are the same.
[0362] In one embodiment, HX.sup.1 and HX.sup.2 are different.
[0363] In one embodiment, HX.sup.1 and HX.sup.2 are the same, and
are independently selected from HCl, HBr, and HI. In this case, the
compound (a diamino-phenothiazine compound) may conveniently be
referred to as a "diamino-phenothiazine bis(hydrogen halide)
salt".
[0364] In one embodiment, HX.sup.1 and HX.sup.2 are each HCl. In
this case, the compound may conveniently be referred to as a
"diamino-phenothiazine bis(hydrogen chloride) salt".
[0365] In one embodiment, HX.sup.1 and HX.sup.2 are each HBr. In
this case, the compound may conveniently be referred to as a
"diamino-phenothiazine bis(hydrogen bromide) salt".
[0366] In one embodiment, HX.sup.1 and HX.sup.2 are each HI. In
this case, the compound may conveniently be referred to as a
"diamino-phenothiazine bis(hydrogen iodide) salt".
Some Preferred Combinations
[0367] In one embodiment:
[0368] each of R.sup.1 and R.sup.9 is independently --H, -Me, or
-Et; and
[0369] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2.
[0370] In one embodiment:
[0371] each of R.sup.1 and R.sup.9 is independently --H, -Me, or
-Et; and
[0372] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2.
[0373] In one embodiment:
[0374] each of R.sup.1 and R.sup.9 is independently --H; and
[0375] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2.
[0376] In one embodiment:
[0377] each of R.sup.1 and R.sup.9 is independently --H; and
[0378] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2.
[0379] In one embodiment:
[0380] each of R.sup.1 and R.sup.9 is independently --H, -Me, or
-Et; and
[0381] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2; and
[0382] each of HX.sup.1 and HX.sup.2 is independently selected from
HCl, HBr, and HI.
[0383] In one embodiment:
[0384] each of R.sup.1 and R.sup.9 is independently --H, -Me, or
-Et; and
[0385] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
[0386] each of HX.sup.1 and HX.sup.2 is independently selected from
HCl, HBr, and HI.
[0387] In one embodiment:
[0388] each of R.sup.1 and R.sup.9 is independently --H; and
[0389] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2 or
--NEt.sub.2; and
[0390] each of HX.sup.1 and HX.sup.2 is independently selected from
HCl, HBr, and HI.
[0391] In one embodiment:
[0392] each of R.sup.1 and R.sup.9 is independently --H; and
[0393] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
[0394] each of HX.sup.1 and HX.sup.2 is independently selected from
HCl, HBr, and HI.
[0395] In one embodiment:
[0396] each of R.sup.1 and R.sup.9 is independently --H; and
[0397] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
[0398] each of HX.sup.1 and HX.sup.2 is HCl.
##STR00008##
[0399] In one embodiment:
[0400] each of R.sup.1 and R.sup.9 is independently --H; and
[0401] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and
[0402] each of HX.sup.1 and HX.sup.2 is HBr.
##STR00009##
[0403] In one embodiment:
[0404] each of R.sup.1 and R.sup.9 is independently --H; and
[0405] each of the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) is independently --NMe.sub.2; and each of
HX.sup.1 and HX.sup.2 is HI.
##STR00010##
Isotopic Variation
[0406] In one embodiment, one or more of the carbon atoms of the
compound is .sup.11C, .sup.13C, or .sup.14C.
[0407] In one embodiment, one or more of the carbon atoms of the
compound is .sup.11C.
[0408] In one embodiment, one or more of the carbon atoms of the
compound is .sup.13C.
[0409] In one embodiment, one or more of the carbon atoms of the
compound is .sup.14C.
[0410] In one embodiment, one or more of the nitrogen atoms of the
compound is .sup.15N.
[0411] In one embodiment, one or more or all of the carbon atoms of
one or more or all of the groups R.sup.3NA, R.sup.3NB, R.sup.7NA,
R.sup.7NB, R.sup.1, R.sup.9, and R.sup.10 is .sup.11C. (Or
.sup.13C.) (Or .sup.14C.)
[0412] In one embodiment, one or more or all of the carbon atoms of
one or more or all of the groups R.sup.3NA, R.sup.3NB, R.sup.7NA
and R.sup.7NB is .sup.11C. (Or .sup.13C.) (Or .sup.14C.)
[0413] In one embodiment, the groups --N(R.sup.3NA)(R.sup.3NB) and
--N(R.sup.7NA)(R.sup.7NB) are the same, and are:
--N(.sup.11CH.sub.3).sub.2. (Or --N(.sup.13CH.sub.3).sub.2.) (Or
--N(.sup.14CH.sub.3).sub.2.)
[0414] In one embodiment, the compound is selected from the
following compounds, and pharmaceutically acceptable salts,
solvates, and hydrates thereof.
##STR00011##
Other Aspects of the Invention
[0415] Where any method of treatment is disclosed herein, also
disclosed are:
[0416] A DAPTZ compound for use in that method and use of DAPTZ
compound in the preparation of a medicament for said treatment.
Corresponding embodiments, preferences, and individualizations,
described herein apply mutatis mutandis to these aspects.
[0417] Thus the invention provides inter alia:
[0418] A DAPTZ compound for use in a method of treatment of a
cognitive or CNS disorder in a patient, wherein said disorder is
one which is susceptible to treatment by said DAPTZ compound, which
method comprises orally administering to said patient a dosage unit
containing said DAPTZ compound in oxidised form as active
ingredient, wherein said dosage unit releases at least 50% of said
active ingredient within 30 minutes under standard conditions.
[0419] A DAPTZ compound for use in a method of treatment of a
cognitive or CNS disorder in a patient, wherein said disorder is
one which is susceptible to treatment by said DAPTZ compound, which
method comprises orally administering to said patient a dosage unit
containing said DAPTZ compound in oxidised form as active
ingredient, wherein said dosage unit is gastroretained.
[0420] A DAPTZ compound for use in a method of treatment of a
cognitive or CNS disorder in a patient, wherein said disorder is
one which is susceptible to treatment by said DAPTZ compound, which
method comprises orally administering to said patient a dosage unit
containing said DAPTZ compound in stable crystalline reduced form
as active ingredient,
[0421] Use of a DAPTZ compound in the preparation of a medicament
dosage unit for use in a method of treatment of a cognitive or CNS
disorder in a patient, wherein said disorder is one which is
susceptible to treatment by said DAPTZ compound, which method
comprises orally administering to said patient said dosage unit
containing said DAPTZ compound in oxidised form as active
ingredient, wherein said dosage unit releases at least 50% of said
active ingredient within 30 minutes under standard conditions.
[0422] Use of a DAPTZ compound in the preparation of a medicament
dosage unit for use in a method of treatment of a cognitive or CNS
disorder in a patient, wherein said disorder is one which is
susceptible to treatment by said DAPTZ compound, which method
comprises orally administering to said patient said dosage unit
containing said DAPTZ compound in oxidised form as active
ingredient, wherein said dosage unit is gastroretained.
[0423] Use of a DAPTZ compound in the preparation of a medicament
dosage unit for use in a method of treatment of a cognitive or CNS
disorder in a patient, wherein said disorder is one which is
susceptible to treatment by said DAPTZ compound, which method
comprises orally administering to said patient a dosage unit
containing said DAPTZ compound in stable crystalline reduced form
as active ingredient,
[0424] In one aspect the invention provides a drug unit for the
treatment of a cognitive or CNS disorder in a patient, wherein said
disorder is one which is susceptible to treatment by a DAPTZ
compound, which dosage unit contains said DAPTZ compound in
oxidised form as active ingredient, and wherein said dosage unit
releases at least 50% of said active ingredient within 30 minutes
under standard conditions. The dosage units comprise may comprise,
for example, 40, 45, 50, 55, 60, 65, 70, 100, 120 mg of a DAPTZ
compound as described.
[0425] In one aspect the invention provides a drug unit for the
treatment of a cognitive or CNS disorder in a patient, wherein said
disorder is one which is susceptible to treatment by a DAPTZ
compound, which dosage unit contains said DAPTZ compound in
oxidised form as active ingredient, and wherein said dosage unit is
gastroretained. The dosage unit may comprisee at least 50, 60, 70,
80, 90 or 100 mg, or more e.g. 200, 300, 400, 500 mg of a DAPTZ
compound as described.
[0426] In one aspect the invention provides a drug unit for the
treatment of a cognitive or CNS disorder in a patient, wherein said
disorder is one which is susceptible to treatment by a DAPTZ
compound, which dosage unit contains said DAPTZ compound in stable
crystalline reduced form as active ingredient, at a dosage
described above (e.g. 100, 200, 300, 400, 500, 600, 700, 800, 900,
or 1000 mg), and having a dissolution rate described above (e.g.
<50% in 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours).
[0427] Also provided is a drug product comprising said unity
accompanied by a label indicating that the drug product is for the
treatment of said disease, the container containing one or more
dosage units each comprising at least one pharmaceutically
acceptable excipient and, as an active ingredient, an isolated pure
diaminophenothiazinium compound as described herein.
Ligands
[0428] Additionally use as diagnostic or prognostic indicator e.g.
as a ligand for labelling protein aggregates in the brain is also
contemplated. The findings herein in which cognitive effect
(dependent on brain concentration) vs. negative haematological
effect (deduced to be from dimer formation) have implications also
for use as a ligand.
[0429] Such DAPTZ compounds (ligands) may incorporate, be
conjugated to, be chelated with, or otherwise be associated with,
other chemical groups, such as stable and unstable detectable
isotopes, radioisotopes, positron-emitting atoms, magnetic
resonance labels, dyes, fluorescent markers, antigenic groups,
therapeutic moieties, or any other moiety that may aid in a
prognostic, diagnostic, or therapeutic application.
[0430] For example, in one embodiment, the DAPTZ compound is as
defined herein, but with the additional limitation that the
compound incorporates, is conjugated to, is chelated with, or is
otherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)
detectable labels, for example, isotopes, radioisotopes,
positron-emitting atoms, magnetic resonance labels, dyes,
fluorescent markers, antigenic groups, or therapeutic moieties.
[0431] In one embodiment, the DAPTZ compound is a ligand as well as
a label, e.g., a label for tau protein (or aggregated tau protein),
and incorporates, is conjugated to, is chelated with, or is
otherwise associated with, one or more (e.g., 1, 2, 3, 4, etc.)
detectable labels.
[0432] For example, in one embodiment, the DAPTZ compound is as
defined above, but with the additional limitation that the compound
incorporates, is conjugated to, is chelated with, or is otherwise
associated with, one or more (e.g., 1, 2, 3, 4, etc.) detectable
labels.
[0433] Labelled DAPTZ compounds (e.g., when ligated to tau protein
or aggregated tau protein) may be visualised or detected by any
suitable means, and the skilled person will appreciate that any
suitable detection means as is known in the art may be used.
[0434] For example, the DAPTZ compound (ligand-label) may be
suitably detected by incorporating a positron-emitting atom (e.g.,
.sup.11C) (e.g., as a carbon atom of one or more alkyl group
substituents, e.g., methyl group substituents) and detecting the
compound using positron emission tomography (PET) as is known in
the art.
[0435] Such .sup.11C labelled DAPTZ compounds may be prepared by
adapting the methods described herein in known ways, for example,
in analogy to the methods described in WO 02/075318 (see FIGS. 11a,
11b, 12) and WO 2005/030676.
[0436] Thus in one aspect there is disclosed a method of labelling
an aggregated disease protein associated with a neurodegenerative
disorder in the brain of a patient, wherein said aggregated disease
protein is one which is susceptible to labelling by a DAPTZ
compound, [0437] which method comprises orally administering to
said patient a dosage unit containing said DAPTZ compound in
oxidised form as active-labelled ingredient, [0438] wherein said
dosage unit releases at least 50% of said active ingredient within
30 minutes under standard conditions.
[0439] In a further aspect there is disclosed a method of labelling
an aggregated disease protein associated with a neurodegenerative
disorder in the brain of a patient, wherein said aggregated disease
protein is one which is susceptible to labelling by a DAPTZ
compound, [0440] which method comprises orally administering to
said patient a dosage unit containing said DAPTZ compound in
oxidised form as active-labelled ingredient, [0441] wherein said
dosage unit is gastroretained.
[0442] In a further aspect there is disclosed a method of labelling
an aggregated disease protein associated with a neurodegenerative
disorder in the brain of a patient, wherein said aggregated disease
protein is one which is susceptible to labelling by a DAPTZ
compound, [0443] which method comprises orally administering to
said patient a dosage unit containing said DAPTZ compound in stable
crystalline reduced form as active-labelled ingredient.
[0444] Preferred aggregated disease proteins, DAPTZ compounds, and
neurodegenerative disorders are discussed elsewhere herein.
[0445] The methods may further comprise the step of determining the
presence and/or amount of said compound bound to said aggregated
protein. Another aspect of the present invention pertains to a
method of diagnosis or prognosis of said neurodegenerative disorder
which further comprises the step of correlating the result of the
determination with the disease state of the subject.
[0446] Where any method of labelling, diagnosis or prognosis is
disclosed herein, also disclosed are:
[0447] A DAPTZ compound for use in that method and use of DAPTZ
compound in the preparation of a diagnostic or prognostic indicator
for said method. Corresponding embodiments, preferences, and
individualizations, described herein apply mutatis mutandis to
these aspects.
[0448] Throughout this specification, including the claims which
follow, unless the context requires otherwise, the word "comprise,"
and variations such as "comprises" and "comprising," will be
understood to imply the inclusion of a stated integer or step or
group of integers or steps but not the exclusion of any other
integer or step or group of integers or steps.
[0449] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a pharmaceutical carrier" includes
mixtures of two or more such carriers, and the like.
[0450] Ranges are often expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by the use of the
antecedent "about," it will be understood that the particular value
forms another embodiment.
[0451] All compatible combinations of the embodiments described
above are explicitly disclosed herein as if each combination was
specifically and individually recited.
[0452] Any sub-titles herein are included for convenience only, and
are not to be construed as limiting the disclosure in any way.
[0453] The invention will now be further described with reference
to the following non-limiting Figures and Examples. Other
embodiments of the invention will occur to those skilled in the art
in the light of these.
[0454] The disclosure of all references cited herein, inasmuch as
it may be used by those skilled in the art to carry out the
invention, is hereby specifically incorporated herein by
cross-reference.
FIGURES
[0455] FIG. 1. TRx-014-001 & 009 clinical trial study design.
The numbers correspond to the patients at each stage of the study.
323 patients entered the base study, one subject was randomised but
not given medication. Subjects were treated with MTC as indicated
or placebo. After 24 weeks and 50 weeks, subjects continued into 2
extensions (E1 and E2) of the trial and then continued in trial
TRx-014-009. For ethical reasons, those on placebo for the first 24
weeks were given 100 mg bd in E1. "tid" means dosing at a frequency
of three times per day, and "bd" means dosing at a frequency of
twice per day.
[0456] FIG. 2. Treatment response in CDR-moderates at 24 weeks. For
this chart, the labelling conventions of "plac" refers to placebo,
"low" refers to low (100 mg) (see footnote 1, Table 1) "30 mg"
refers to 30 mg dose tid and "60 mg" refers to 60 mg dose tid.
[0457] FIG. 3. Comparison of treatment effects of Rember.TM. as
seen by functional brain imaging using SPECT. Decreased regional
cerebral blood flow (rCBF) is seen as areas of white across the
brain.
[0458] (1) SPM analysis shows regions where visit 4 had
significantly less rCBF than visit 1 in subjects treated with
placebo. Threshold for difference p<0.005, corrected p<0.05
for multiple comparisons, both voxel and cluster significance.
R=right, L=left, A=anterior, P=posterior. The upper pair in each
panel represent anterior (left) and posterior (right) views
respectively.
[0459] (2) SPM analysis shows no regions where visit 4 had
significantly less rCBF than visit 1 in subjects treated with
Rember.TM. at 30 mg or 60 mg tid. Threshold for difference
p<0.005, corrected p<0.05 for multiple comparisons, both
voxel and cluster significance.
[0460] (3) Locations of treatment-dependent difference in decline
between baseline and visit 4 in CDR-mild subjects treated with
placebo versus those with Rember.TM. at 30/60 mg tid. Threshold for
difference p<0.005, corrected p<0.05 for multiple
comparisons, both voxel and cluster significance.
[0461] FIG. 4. ITT/OC ADAS-cog change from baseline and fitted
curves. For this chart, the labelling conventions of "placlow"
refers to subjects who were originally randomised to placebo and
were then switched to the 100 mg dose bd after 24 weeks, "low"
refers to low (100 mg) dose tid, "30 mg" refers to 30 mg dose tid
and "60 mg" refers to 60 mg dose tid.
[0462] FIG. 5. Dissolution of capsules in simulated intestinal
fluid by dosage: (A) 30 mg and (B) 100 mg capsules dissolved
initially and following 24 months storage. Dissolution of the 100
mg capsule was slower than the 30 mg capsule and this difference
increased with time since manufacture.
[0463] FIG. 6A. Rember.TM. inhibits nucleation event and
autocatalytic tau aggregation.
[0464] FIG. 6B. Rember.TM. opens a new clearance pathway for tau
aggregates.
[0465] FIG. 7. Relationship between dissolution time and relative
cognitive and haematological effects. Dissolution % is .alpha.
adjusted based on the calculations below:
[0466] A cognitive activity index (CI) was first determined as the
normalised ADAS-cog effect size at 50 weeks at each nominal dose
relative to the maximal effect size observed at 50 weeks, using the
linear least-squares estimates of effect size. A corresponding
haematological activity index (HI) was expressed as the normalised
change in red-cell count at 24 weeks at each nominal dose relative
to the maximal red cell effect size observed. The time-points of 50
weeks for cognitive and 24 weeks for haematological effects were
chosen because the corresponding effects were maximal at these
times. The relative cognitive activity was expressed in the form
CI/(CI+HI) and the relative haematological activity was expressed
in the form HI/(CI+HI), and both of these relative activities were
normalised to their corresponding maxima across doses. A similar
calculation was used to express the relative percentage of MTC
available in solution before or after 30 minutes relative to the
total, based on dissolution data from 24-month-old capsules, when
the dissolution differences between capsule strengths were maximal.
The relationships explicitly calculated can be expressed as
follows:
.alpha.CI/[.alpha.CI+(1-.alpha.)HI]{tilde over
(=)}D.sub.30/D.sub.total (1)
(1-.alpha.)HI/[.alpha.CI+(1-.alpha.)HI]{tilde over
(=)}(1-D.sub.30)/D.sub.total (2)
where .alpha. is a scaling parameter for relating CI units to HI
units (found to be 0.645 by least squares estimation), D.sub.30 is
the percentage of total MTC available from 24-month capsules at 30
minutes, and D.sub.total is the total nominal dose which is
eventually dissolved.
[0467] FIG. 8. Implied dose-response relationship at 50 weeks.
Effect sizes calculated using linear least-squares estimates at 50
weeks. The effective therapeutic dose available from the 100 mg
capsule was equivalent to a dose of approximately 25 mg, indicating
that the capsules did not permit proportionate delivery and
absorption of the nominal dose in a therapeutically active
form.
[0468] FIG. 9. Differences in key red cell parameters in rats
between MTC and L-MTx administered orally for 14-days at the
indicated daily doses. Differences are shown terms of change
observed with L-MTx with respect to MTC. For example, at a dose of
150 mg/kg L-MTx, red cell count is increased by
>1.times.10.sup.6/.mu.l and the mean cell haemoglobin
concentration is increased by 3 pg/dL. The statistical analysis of
the data is shown in Table 4. Abbreviations and units: RBC: red
cell count, 10.sup.6/.mu.L; HB: haemoglobin, g/dL; MCV: mean cell
volume, fL; MCHC: mean cell haemoglobin concentration, g/dL; RETI:
reticulocyte count, % of red cells.
[0469] FIG. 10. Average urinary excretion rates for oxidised-MTC
(Ox-MT) from 7 adult human subjects following 10 mg oral dose
(mean, SE). (From DiSanto and Wagner, 1972b).
[0470] FIG. 11. Average urinary excretion rates for leuco-MT (L-MT)
from 7 adult human subjects following 10 mg oral dose (mean, SE).
(From DiSanto and Wagner, 1972b).
[0471] FIG. 12. Urinary excretion rate for Ox-MTC following a 10 mg
oral dose.
[0472] FIG. 13. Urinary excretion rate for L-MT following a 10 mg
oral dose.
[0473] FIG. 14. Concentration of Ox-MT in whole blood after
intravenous administration of 100 mg MTC (from Peter et al.,
2000).
[0474] FIG. 15. Concentration of Ox-MT in whole blood after oral
administration of 100 mg MTC with (open circles) or without (filled
circles) 800 mg of Mesna (mean, SE) (from Peter et al., 2000).
[0475] FIG. 16. Estimation of apparent bioavailability based on
excretion of total-MT (i.e. Ox-MT+L-MT) at T-infinity following
oral dosing, where the curve has been fitted by the empirical
equation:
Urinary recovery=88.9-(88.9.times.Dose)/(69.7+Dose)
Note the lower than expected value (marked "P") for the 100 mg dose
result reported by Peter et al. (corrected for expected 48-hr
excretion).
[0476] FIG. 17. Rate of urinary excretion of total MTC (.mu.mol/h)
during the indicated time intervals after i.v. (black bars) and
oral (grey bars) administration of MTC. Mean, SE, n=7 (from Peter
et al., 2000).
[0477] FIG. 18. First stage of model: fitting blood concentration
data following single intravenous dose of 100 mg MTC and scaled
urinary excretion data following single oral dose of 10 mg MTC.
[0478] FIG. 19. Fit between observed blood concentration data
following intravenous dosing from Peter et al. (Table 7) and
prediction of the model depicted in FIG. 18.
[0479] FIG. 20. Fit between scaled observed urinary excretion of
Ox-MT following a single oral dose of 10 mg MTC from DiSanto and
Wagner (Table 5) and prediction of the model (shown in FIG. 18)
after single intravenous dose of MTC (100 mg).
[0480] FIG. 21. Fit between scaled observed urinary excretion of
L-MT following single oral dose of 10 mg MTC from DiSanto and
Wagner (Table 5) and prediction of the model (shown in FIG. 18)
after single intravenous dose of MTC (100 mg).
[0481] FIG. 22. Second stage of model: fitting blood concentration
data following a single oral dose of 100 mg MTC and scaled urinary
excretion data following a single oral dose of 10 mg MTC.
[0482] FIG. 23. Fit between observed blood concentration data
following oral dosing from Peter et al. (Table 7) and prediction of
the model depicted in FIG. 22.
[0483] FIG. 24. Fit between scaled observed urinary excretion of
Ox-MT following single oral dose of 10 mg MTC from DiSanto and
Wagner (Table 5) and prediction of the model (shown in FIG. 22)
after single oral dose of MTC (100 mg).
[0484] FIG. 25. Fit between scaled observed urinary excretion of
L-MT following single oral dose of 10 mg MTC from DiSanto and
Wagner (Table 5) and prediction of the model (shown in FIG. 22)
after single oral dose of MTC (100 mg).
[0485] FIG. 26. Comparison of mean urinary excretion rates of total
MT as reported by Peter et al. and those predicted by the oral
model shown in FIG. 22 for the same intervals. Comparison of total
excretion over 24 hr is shown.
[0486] FIG. 27. This reproduces FIG. 16, but includes the model
prediction ("M") for excretion of MTC. The model value is closer to
that predicted from other studies than the estimate reported by
Peter et al. ("P").
[0487] FIG. 28. Outputs of the oral model for C2 (blood), C4 and C3
are shown rescaled to their corresponding maxima. These are
compared with a triexponential model applied to the measured level
of MT in pig brain following a single oral dose.
[0488] FIG. 29. Relationship between observed clinical efficacy of
Rember.TM. and predicted average steady state level of MT in C3 for
the 3/day dosing regime. Also shown are the predicted steady state
levels of MT in C3 for 2/day and 1/day dosing regimes.
[0489] FIG. 30. Relationship between observed clinical efficacy of
Rember.TM. and predicted average steady state level of MT in C2 for
the 3/day dosing regime. Also shown are the predicted steady state
levels of MT in C2 for 2/day and 1/day dosing regimes.
[0490] FIG. 31A. Difference between observed effect size and
predicted effect size as a function of percent capsule dissolution
at 30 minutes. Capsule dissolution is determined by the amount of
MTC released into the aqueous phase in standard US/EU Pharmacopoeia
dissolution conditions.
[0491] FIG. 31B. Relationship between expected steady-state level
of MT in the central compartment (C2, i.e. blood) and observed loss
of red cells at 24 expressed (expressed as fractional change
relative to normal range).
[0492] FIG. 32. Relationship between actual dose ("dose") and
effective dose ("eff dose") based on urinary excretion data.
[0493] FIG. 33. Comparison of predicted fraction absorbed for MTC
and L-MTx assuming that administration of the L-MTx form eliminates
non-absorption from the stomach (ie C1 in FIG. 22).
[0494] FIG. 34. Relationship between expected clinical efficacy of
an L-MTx-based form of the methylthioninium moiety and predicted
average steady state level of MT in C3 for a range of dosing
regimes from 1/day to 3/day.
[0495] FIG. 35. Relationship between expected clinical efficacy of
an L-MTx-based form of the methylthioninium moiety and predicted
average steady state level of MT in C2 (blood) for a range of
dosing regimes from 1/day to 3/day.
[0496] FIG. 36. Observed dose-response relationship for effect of
MTC in the capsule formulation used in the trial TRx-014-001 on
loss of red cells and for MTC-based and expected dose-response
relationship for an L-MTx-based form of a methylthioninium
medicinal product administered at the doses indicated at a
frequency of 3/day.
[0497] FIG. 37. Various quantitative models for the progression and
treatment of Alzheimer's Disease as described in Example 12.
[0498] FIG. 38. Relationship between expected clinical efficacy of
an L-MTx-based preparation for 1/day slow-release formulation.
[0499] FIG. 39. The differential effect of inhibitors of different
sites of the tau aggregation pathway. The scheme on the left shows
the site of inhibition of tau entry into the tau aggregation
pathway (input) and the site of enhanced clearance of tau
aggregates from that pathway. The effect of changes at both of
these two sites on PHF levels in neurons is shown in the right
panel. Inhibition of input decreases the level of PHFs initially,
before the rate of formation continues at the same level as before.
Enhanced clearance of aggregated tau, however, results in a steady
decrease in the level of aggregated tau.
[0500] FIG. 40. Tau aggregation and its clearance in Alzheimer's
disease. Tau oligomers can either assemble into filamentous PHFs
and/or enter the endosomal-lysosomal clearance pathway.
EXAMPLES
Example 1
Phase 2 Clinical Trial TRx-014-001
Summary
[0501] A 50-week Phase 2 exploratory dose-range-finding study for
treatment of mild and moderate dementia of the Alzheimer type has
been conducted using an investigational medicinal product (IMP) of
which MTC was the active pharmaceutical ingredient (API). The study
was a randomized, double blinded, placebo-controlled study whose
primary objective was to investigate the effects of MTC at three
doses (30, 60 and 100 mg, each three times pe'r day) compared with
placebo on cognitive ability (as measured by the ADAS-cog scale:
Alzheimer's Disease Assessment Scale--cognitive subscale). There
were 322 subjects randomized, of whom 245 (74%) completed the first
24 weeks of treatment. Of these, 227 (93%) chose to continue
treatment for a further 6 months, of whom 177 (78%) completed 50
weeks of treatment on 2 Jul. 2007. The final analyses comprise
analyses of the ITT/OC (Intention to Treat/Observed Case)
population of 245 subjects who completed 24 weeks of treatment, and
177 subjects who completed 50 weeks of treatment by 2 Jul. 2007.
The study design is summarized in FIG. 1. For reasons of ethical
concerns, subjects who were originally randomized to placebo during
the first 6-month phase were switched to the 100 mg dose during the
second 6-month extension phase of the study ("E1").
24-week Analyses
[0502] The primary pre-specified outcome was an ITT/OC analysis of
ADAS-cog change from baseline at 24 weeks using an analysis of
covariance approach which included an assessment of the interaction
between the effect of treatment with Rember.TM. and baseline
severity as defined by CDR (Clinical Dementia Rating scale). This
analysis demonstrated a positive effect of Rember.TM. at 60 mg tid
which achieved statistical significance in both the ITT/OC and
ITT/LOCF (Intention to Treat/Last Observation Carried Forward)
populations. CDR severity at baseline was found to be a highly
significant cofactor, and when included in the model showed that
the effect of Rember.TM. was significant at 24 weeks only in
subjects who were CDR-moderate at baseline. The lack of decline in
CDR-mild subjects on placebo prevented efficacy analysis in this
group over the first 24 weeks. However Rember.TM.'s efficacy was
confirmed in this group by functional brain scan analysis at 24
weeks, and by ADAS-cog at 50-weeks.
TABLE-US-00001 TABLE 1 ADAS-cog effect size at 24 weeks in
CDR-moderates (in ADAS-cog units) Dose.sup.(1) Estimate 95% CI
p-value.sup.(2) low (100 mg) -0.42 -4.24, 3.40 0.826 30 mg -4.02
-7.30, -0.74 0.0172 60 mg -5.41 -9.31, -1.52 0.0073 .sup.(1)The 100
mg dose is referred to as the "low (100 mg)" dose to indicate that
in its present formulation, the therapeutic efficacy of the 100 mg
capsule did not correspond to the nominal dose. .sup.(2)The p-value
is from a test of whether the value is significantly different from
placebo.
[0503] In the analysis of the subgroup of the ITT/OC population who
were CDR-moderate at baseline (FIG. 2), the effect size of
Rember.TM. at the 60 mg tid dose was -5.4 ADAS-cog units and 3.4
MMSE (Mini-Mental State Examination) units (MMSE data not shown).
Whereas placebo-treated subjects declined by 5.1 ADAS-cog units,
there was no evidence of decline in subjects treated with
Rember.TM. at 30 mg or 60 mg tid over 24 weeks. Non-cognitive
outcome variables (measuring psychiatric disturbance and activities
of daily living skills) also confirmed the disease-stabilising
properties and efficacy size of Rember.TM. in the moderate group.
Subjects receiving Rember.TM. at the 60 mg tid dose, showed an
effect size of 1.4-1.9 units on the CGIC (Clinical Global
Impression of Change) scale at 24 weeks relative to placebo,
registered by clinical assessors blinded to the other outcome
measures. The odds-ratio of not declining on CGIC for subjects
taking Rember.TM. at the 60 mg dose was 9 times better than
placebo. The CDR-sum-of-boxes parameter, another global clinical
measure, showed benefit of -1.7 units. Finally, Rember.TM. at the
60 mg dose showed significant benefit on the ADFACS (Alzheimer's
Disease Functional Assessment Scale) measure of activities of daily
living, with an effect size of 3.1 to 6.1 units over 24 weeks. In
all the psychometric analyses at 24 weeks, the 100 mg capsule
showed minimal efficacy, consistent with a formulation defect of
the capsules at this dosage strength discussed further below.
[0504] The 100 mg dose is referred to as the "low (100 mg)" dose to
indicate that in its present formulation, the therapeutic efficacy
of the 100 mg capsule did not correspond to the nominal dose. This
is discussed in more detail in the Examples below.
Functional Brain Scan Analysis
[0505] Prevention of decline over 24 weeks was independently
confirmed by analysis of functional brain scan changes in 135
subjects who had undergone two SPECT scans 6 months apart on
average (FIG. 3). Whereas subjects receiving placebo showed the
expected pattern of deterioration in frontal and temporo-parietal
regions of the brain, subjects receiving Rember.TM. at 30 mg or 60
mg showed no evidence of deterioration in any brain region. When
the subgroup who were CDR-mild at baseline were examined
separately, there was also evidence of prominent decline over 6
months in subjects receiving placebo, amounting to loss of 8% of
functioning neuronal volume. The treatment effect seen in the whole
population was also seen in the CDR-mild subgroup, demonstrating
the efficacy of Rember.TM. in CDR-mild AD. The fact that there was
objective evidence of progressive functional deterioration in the
mild subgroup without corresponding evidence of decline on any of
the psychometric scales over 6 months confirms the powerful
confounding influence of cognitive reserve in mild AD. Overall,
despite this effect, baseline functional deficits shown by SPECT
scan were highly correlated with baseline ADAS-cog score, and the
benefit of treatment with Rember.TM. shown on the ADAS-cog scale
was likewise correlated with the functional benefit demonstrated by
SPECT scan. Rember.TM.'s action seen by functional brain scan
strongly suggests that Rember.TM.'s ability to reverse the Tau
aggregation pathology, which is known to occur in the same brain
regions as those showing functional brain scan defects, is
responsible for its ability to prevent decline in cortical brain
function in the same regions. Given the greater sensitivity of
SPECT in detection of both decline and treatment effects, and its
ability to predict treatment response (see 50-week analysis below),
it is concluded that SPECT could be used as a surrogate or proxy
marker for future clinical trials aiming to demonstrate disease
modification.
50-Week Analyses
[0506] The 50-week study extended and confirmed the findings of the
24-week study, and demonstrated significant benefits in both
CDR-mild and CDR-moderate subjects in the overall ITT/OC and
ITT/LOCF populations (FIG. 4; Tables 2 and 3). Subjects originally
randomized to placebo were switched to the low (100 mg) dose bd
after 24 weeks. This is referred to as the "placebo-low" treatment
arm. Because of the minimal efficacy of the low (100 mg) dose on
any of the psychometric scales over the first 24 weeks of
treatment, the placebo-low treatment arm conveniently served as the
Least Exposed Dose comparator arm for the 50-week study.
[0507] The mean decline observed over the 50-week study in
placebo-treated subjects was 7.8 ADAS-cog units (FIG. 4). For
subjects treated with Rember.TM. at a dose of 60 mg tid, the
decline seen over 50 weeks was not significantly different from
zero on either the ADAS-cog scale or the MMSE scale for subjects.
On the ADAS-cog scale, about 60% of subjects improved or stayed the
same at 50 weeks. On the MMSE scale, 62% improved or stayed the
same at 50 weeks. The odds of a patient not declining on either
scale were about 3.4 times better at the 60 mg dose than on
placebo-low. The corresponding effect sizes were -6.8 ADAS-cog
units and 3.2 MMSE units over the 50-week trial. In addition to the
effect on disease progression, there was an initial symptomatic
improvement at 15 weeks of 1.6 ADAS-cog units and 0.8 MMSE units at
the 60 mg dose, comparable to that observed with AChE
inhibitors.
TABLE-US-00002 TABLE 2 Effect sizes inferred from mixed effects
analysis at 50 weeks (in ADAS-cog units) Dose Estimate 95% CI
p-value.sup.(1) low (100 mg) -4.04 -7.21, -0.87 0.0124 30 mg -3.87
-6.90, -0.84 0.0126 60 mg -6.78 -9.74, -3.82 <0.0001 .sup.(1)The
p-value is from a test of whether the value is significantly
different from placebo.
TABLE-US-00003 TABLE 3 Effect sizes inferred from least-squares
analysis at 50 weeks (in ADAS-cog units) Dose Estimate 95% CI
p-value.sup.(1) low (100 mg) -3.59 -5.81, -1.37 0.0015 30 mg -4.37
-6.83, -1.92 0.0005 60 mg -6.50 -8.89, -4.14 <0.0001 .sup.(1)The
p-value is from a test of whether the value is significantly
different from placebo.
[0508] There was no deterioration on the non-cognitive scales in
CDR-mild subjects in the placebo-low arm over 50 weeks. The
non-cognitive outcomes at 50 weeks in CDR-moderate subjects
confirmed the findings of the 24-week analyses. The NPI
(Neuropsychiatric Inventory) demonstrated benefits for Rember.TM.
treatment over 50 weeks. Whereas subjects in the placebo-low arm
declined by 9.6 units on the patient-disturbance scale and 4.9
units on the carer-distress scale, no such decline was seen in
subjects continuously treated with Rember.TM. over 50 weeks, with
corresponding best effect sizes of -9.2 units and -4.6 units.
[0509] The placebo-low arm compared to the low (100 mg) arm
provided a close approximation to a delayed start design to confirm
that Rember.TM. is disease modifying in a formal regulatory sense.
Subjects who began later on a dose of minimal apparent therapeutic
efficacy as judged by ADAS-cog over the initial 24 weeks remained
significantly different at 50 weeks relative to subjects who had
been receiving the low (100 mg) dose continuously. Furthermore
subjects treated continuously at the low (100 mg) dose showed
retardation in the rate of disease progression. Although there was
a difference in the capsule dosage regime between the two arms (tid
vs. bd), haematological side effects, which showed a clear
dose-response profile, were indistinguishable with regard to the
two dosing regimes, supporting the approximate equivalence of
biological exposure, and hence supporting the inference that
Rember.TM. is disease-modifying. This is also confirmed by
Rember.TM.'s ability to arrest disease progression over 50 weeks at
the 60 mg dose, and reduced the rate of disease progression at the
30 mg and low (100 mg) doses at 50 weeks.
Summary of Clinical Safety of Rember.TM.
[0510] The overall adverse event profile was substantially better
in the Rember.TM. trial than for AChE (Acetylcholine Esterase)
inhibitors at optimal treatment dose reported in the Cochrane
Review (Birks, 2006). There were no significant differences in the
odds of subjects taking Rember.TM. at 30 mg or 60 mg tid
withdrawing, experiencing any adverse event or withdrawing due to
an adverse event, compared with AChE inhibitors. Diarrhoea was the
most frequent adverse event reported by subjects treated with
Rember.TM., particularly the low (100 mg) dose, most likely due to
transit of non-absorbed Rember.TM. to the distal bowel, causing
repopulation of gut flora due to a mild antibiotic activity of MTC
which has been well documented in literature (Kristiansen and
Amaral, 1997; Gunics et al., 2000). Although subjects receiving
Rember.TM. had higher odds of developing diarrhea than reported for
AChE inhibitors, subjects taking Rember.TM. reported significantly
less nausea, vomiting, anorexia and abdominal pain, headache,
fatigue and agitation. The experience from some of the trial
centres indicated that diarrhea may be managed with suitable
probiotic preparations (eg dried lactobacillus preparation).
[0511] No changes of clinical significance were seen in any of the
routine clinical chemistry parameters. Small reductions in red-cell
counts, haemoglobin, methaemoglobin and white-cell counts were seen
in subjects treated with Rember.TM., and these changes were
dose-related. The changes were negligible for the 30 mg tid dose,
but became statistically significant for the 60 mg and low (100 mg)
tid doses. In the case of red-cell parameters, they appeared over
24 weeks, but resolved over 50 weeks, except for evidence that the
60 mg tid dose increased methaemoglobin levels at 24 weeks and
stabilized thereafter. At this dose, the mean level of
methaemoglobin increased from the normal mean value of 0.4% to 0.8%
of haemoglobin, but still below the upper limit of normal (1%). In
the case of white-cells, again the changes were negligible for the
30 mg tid dose, but for the 60 mg dose values decreased and then
stabilized at levels not significantly different from the 30 mg
dose over 50 weeks. It is concluded that oxidation of haemoglobin
by an oxidised form of the methylthioninium moiety is the most
likely mechanism responsible for changes in the red cell
parameters.
[0512] Within the period of the study, none of these changes was
clinically significant, and all remained well within the normal
range. Therefore, it is concluded that the changes do not cause
sufficient concern in terms of risk/benefit ratio to impact on the
further clinical development of the 30 mg and 60 mg dosage
strengths. The present formulation of the low (100 mg) dose is not
suitable for further clinical development because of a poorer
efficacy/side-effect profile discussed below.
Example 2
Formulation and Strength of the Investigational Medicinal Product
(IMP)
[0513] The formulation of Rember.TM. used in TRx-014-001 consisted
of Size 1 blue/blue gelatin capsules containing a semisolid fill
comprised of MTC, Gelucire 44/14 and Aerosil 200. Three strengths
of capsule, differing only in fill weight, were manufactured with
target strengths 30, 60 and 100 mg of MTC, respectively. A matching
placebo containing only Gelucire 44/14 was provided. The hard
gelatin capsules and the gelatin used for capsule banding complied
with current guidelines regarding Transmissible Spongiform
Encephalopathies.
[0514] Uniformity of capsules was tested by Appearance, Fill Weight
Uniformity, Assay (modified from USP 27), Chromatographic purity
(TLC as specified by USP 27) and Dissolution using the European
Pharmacopoeia and US Pharmacopoeia rotating paddle method. Six
manufacturing lots of capsules were produced, and were tested for
uniformity and stability.
[0515] Through these dissolution studies, it was found that the
dissolution of the 100 mg capsule in all in vitro conditions was
slower than the 30 mg capsule and that this difference increased
over time since manufacture (FIG. 5). The 60 mg capsule had an
intermediate dissolution profile relative to the 30 mg and 100 mg
data shown in FIG. 5. Further studies have shown that accelerated
cross-linking of the gelatine capsules in the presence of MTC at
high fill-weights (i.e., particularly 100 mg capsules) decreased
the probability of initial capsule breach, although subsequent
dissolution from the breached capsule was rapid. The MTC released
from the capsule was found to retain the expected level of
bio-activity in the in vitro Tau aggregation assay
(WO96/030766).
[0516] This delay in dissolution of the 100 mg capsule is likely to
have shifted the primary site of absorption from the stomach to the
small intestine, leading both to reduced absorption (leading to
diarrhea) and absorption of the majority of the bioavailable dose
as a therapeutically inactive dimeric species. The implied
dose-response relationship discussed further below indicates that
in the present formulation, the equivalent cognitively-active dose
available from the 100 mg capsule was .about.25 mg, when compared
with the cognitive activities of the 30 mg and 60 mg doses.
[0517] The present formulation limits the extent to which higher
doses of Rember.TM. can be explored clinically in future clinical
studies. As discussed further below, there is no theoretical basis
for an efficacy plateau at the 60 mg dose. It is concluded that the
apparent plateau at 60 mg tid reflects a combination of limitations
in solubility, dissolution and absorption of Rember.TM. at higher
dose.
Example 3
Mathematical Efficacy Model
[0518] A kinetic mathematical model has been developed to try to
gain a better understanding of the Tau aggregation process and its
quantitative relationship with cognitive deterioration. The
structure of the model is illustrated below in FIG. 6, showing the
relevant rate constants.
[0519] A broad range of experimental data inputs were used to
derive estimates of the key rate constants in the above model.
These included inter alia: quantitative clinico-pathological
studies linking Tau aggregation and MMSE score in man, estimation
of rate of progression of Braak stages over time (Braak and Braak,
1991), drug dose-response relationship in cell models and in the
Tau binding assay in vitro, drug dose-response relationship in
reduction of Tau pathology in transgenic animals, and a
pharmacokinetic model linking dose to estimated available brain
levels of Rember.TM. in animals and in man discussed further
below.
[0520] The clinical trial data were used to validate this efficacy
model which can in turn explain the relationships between Tau
aggregation, clinical dementia and Rember.TM.'s clinical efficacy
profile. Specifically, no further assumptions implicating the
accumulation of .beta.-amyloid protein or other unknown
neurotransmitter factors are formally required. It is surprising,
given the complexity of the pathophysiology of AD generally assumed
in the field, that an extremely parsimonious set of assumptions and
rate constants can provide the entire basis for a set of formally
definable relationships linking the rate of progression of clinical
dementia, the dynamics of the Tau aggregation cascade illustrated
above and the efficacy of Tau-aggregation inhibitor therapeutic
intervention.
[0521] There are important inferences to be drawn from the model in
explaining Rember.TM.'s mechanism of action. While it appears a
priori, and it is generally assumed in the field, that the
inhibition of the rate of Tau aggregation via the reduction in the
rate of k3 (i.e. inhibition on the input side), would be important
to explain efficacy, this is not borne out by the mathematical
model. The model can be used to show that the impact of a
theoretical drug that acts only on the inflow side of the
aggregation cascade (e.g. strategies to reduce the upstream feed of
products into the stage of aggregated Tau) would produce only a
step-wise transient reduction in Tau aggregation which would be
compensated for over time by continuing aggregation. In other
words, the theoretical impact of such a drug would be only
symptomatic and would not alter the rate of progression of the
disease, even though the mechanism appears to be potentially
disease-modifying because it targets primary pathology. The model
shows that there would still be progressive accumulation of Tau
aggregates over time, and at the same rate as without the drug.
This is primarily because the clearance pathway for the Tau
aggregates remains ineffective in an AD subject and deteriorates
over time at a rate which can be measured by the rate of Braak
stage progression over time. In the case of potential anti-Tau
strategies, this applies particularly to approaches that might be
based on inhibition of Tau phosphorylation, even if Tau
phosphorylation were assumed to be rate-critical for Tau
aggregation, which has been disputed by the inventors (e.g. Wischik
et al., 1997). This further applies to arguments based on the rate
at which .beta.-amyloid protein might, in some as yet unknown
manner, trigger Tau aggregation, as asserted by the recent current
versions of the A.beta. theory of AD pathogenesis (e.g. Selkoe,
2004).
[0522] The most important therapeutic action of Rember.TM. lies in
its ability to enhance the clearance of Tau aggregates by
dissolving the aggregates and releasing previously aggregated Tau
in the form of a monomer which can be processed through a much more
efficient clearance pathway, i.e., the proteasomal pathway. In
terms of the model, the key action of Rember.TM. is to enhance or
open up the rate constant k4b in FIG. 6B. In effect, this opens up
a new, previously unavailable clearance pathway for the Tau
aggregates. This new clearance pathway, the proteasomal clearance
pathway, is depicted by the k4b rate constant in the FIG. 6B.
[0523] The powerful effect of enhanced clearance in the kinetic
model is due to the autocatalytic effect of the aggregates, in that
the rate of aggregation is directly proportional to the aggregate
concentration. This is the primary mechanism responsible for the
long-term predicted change in the rate of disease progression,
which was borne out in the TRx-014-001 clinical trial. The model
raises the possibility that Rember.TM., if given much earlier in
disease progression (i.e., at or even before clinical MCI), could
also modify the structural deterioration in the neuron's clearance
pathway and provide a further rationale for Rember.TM. as a primary
preventive therapy.
[0524] A further feature of the kinetic model is that it would
predict an early symptomatic effect due to initial dissolution of
existing Tau aggregate load. This initial burst of clearance of
existing aggregates is predicted by the model to contribute to an
early symptomatic improvement. This too was borne out in the
Rember.TM. Phase 2 clinical trial.
[0525] The later disease-modifying action of Rember.TM. depends on
the extent to which the ongoing rate of production of Tau
oligomers, and ongoing degradation of the ELM/proteasomal clearance
pathways over time (which is the ultimate determinant of the
inherent rate of progression through the Braak stages over time),
can be neutralised by enhanced clearance due to
solvation/solubilisation of Tau oligomers. Since these factors are
directly proportional to the aggregate concentration, small changes
in the pharmacokinetic profile of the drug can have a large impact
on rate of disease progression. These features of the model were
again borne out by the Phase 2 clinical trial, and emphasise the
need for maximising the bioavailability of the therapeutically
active species that is absorbed. In particular, there is no
inherent mechanism within the model in its present form that would
predict a dose-response plateau.
Example 4
Relationship Between Cognitive and Haematological Activity
[0526] There were defects in the formulation of the 100 mg capsule,
summarised above leading to increasing delay in dissolution over
time since manufacture. Further studies in vitro have shown that
this is most likely due to accelerated cross-linking of the
gelatine capsules in the presence of MTC at high fill-weights
(i.e., 100 mg capsules).
[0527] Published in vitro studies have suggested that absorption of
MTC is a complex process which depends in part on the activity of
an intrinsic cell-surface thiazine-dye reductase activity (Merker
et al., 1998; Merker et al., 2002; May et al., 2004). A
pharmacokinetic ("PK") model (discussed further below) has been
developed based on published studies in humans (DiSanto and Wagner,
1972a,b,c; Peter et al., 2000) which suggests that the half-life of
disappearance of MTC from the primary absorption compartment is 30
minutes, consistent with the stomach being the primary absorption
site for orally ingested MTC.
[0528] MTC is highly ionised when it is in the oxidised form at pH
7 in a non-reducing environment. As such, it has poor lipid
solubility. However, reduction to the reduced ("L-MT") form by
addition of two electrons leads to an uncharged species which is
readily absorbed. In vitro studies suggest that this reduction step
can only occur physiologically at low pH. This property would
explain why the stomach is the most likely primary absorption site.
PK studies in rodents, pig and primate, indicate that the
predominant form of the methylthioninium moiety found in tissues is
the colourless L-MT form, and that after oral administration, only
a small proportion contributes to the oxidised form which can be
readily measured in blood. It is therefore likely that only the
L-MT form can cross the blood-brain barrier, where a new steady
state is established between oxidised and reduced forms within
neurons. After intravenous administration, substantially higher
levels of the oxidised form can be detected in blood than after
oral administration of the same dose (Peter et al., 2000). Further
PK studies in pig have shown that this is due to a difference in
the level of the circulating L-MT form after oral administration,
and not, as suggested by Peter et al., due to poor bioavailability
via the oral route. This suggests that MTC undergoes reduction
during oral absorption and subsequent tissue distribution.
[0529] In circumstances where dissolution was delayed, as for the
100 mg capsule used in the Rember.TM. trial, it is likely that only
limited absorption of the nominal dose could have occurred via the
reductase mechanism which has been described. This would lead to
delayed absorption from the small intestine at higher pH. On the
basis of in vitro studies it is deduced that these circumstances
would favour the formation of a dimer of oxidised MTC monomers
which is well described in literature (Rabinowitch and Epstein,
1941; Lewis et al., 1943; Spencer and Sutter, 1979). Due to
anti-parallel stacking, the dimer has no net charge. Therefore,
delayed dissolution would be expected to lead to delayed absorption
of MTC in the oxidised state at the higher pH of the small
intestine. From in vitro studies, the dimer would not be expected
to have therapeutic activity, but would have haematological effects
due to its ability to oxidise haemoglobin.
[0530] This delayed-dissolution hypothesis is consistent with the
data derived from the Rember.TM. trial. In essence, the trial has
shown that MTC has two systemic pharmacological actions: cognitive
effects and haematological effects. The cognitive effects do not
show a monotonic dose-response relationship, whereas the
haematological effects do (FIG. 8). This suggests that two distinct
species are responsible for the two types of pharmacological
activity: MTC absorbed as the uncharged L-MT form being responsible
for the beneficial cognitive activity, and MTC absorbed as an
oxidised dimeric species being responsible for the oxidation of
haemoglobin. If this were so, it would be expected that a
relationship could be derived linking dissolution time with the two
distinct pharmacological activities at different capsule strengths.
This was indeed found to be the case, as shown in FIG. 8.
[0531] A very high correlation (r=0.996) was found between the
normalised dissolution expressed as percentage dissolved before or
after 30 minutes, and the normalised relative cognitive or
haematological activity indices. For relative dissolution, the
percentage of the total dissolution that occurred in vitro before
or after 30 minutes was calculated. The corresponding partitioning
of total pharmacological activity was derived as shown in FIG.
7.
[0532] It should be borne in mind that the relative cognitive
activity at each nominal dose is expressed as the proportion of
total pharmacological activity (i.e., cognitive and haematological)
at each nominal dose. Therefore, although the 30 mg dose has a
smaller absolute cognitive effect than the 60 mg dose, it has a
higher relative cognitive activity index relative to total
pharmacological activity, because it has less haematological
activity than the 60 mg dose.
[0533] Conversely, the lack of monotonic dose-response relationship
observed in the efficacy analyses of ADAS-cog at 50 weeks implies
that the effective therapeutic dose available from the 100 mg
capsule was as indicated in FIG. 8, i.e., approximately 25 mg, or a
quarter of the nominal dose, similar to the 30 mg dose in activity
at 50 weeks. It is for this reason that in the analyses presented
above, the 100 mg dose was indicated as "low (100 mg)" to signify
that the formulation of these capsules did not permit proportionate
delivery and absorption of the expected nominal dose in its
therapeutically active form. It would appear that a major
determinant of therapeutic activity in the brain is dependent on
absorption in the L-MT form, which may be mediated via ability of
this form to cross the blood-brain barrier.
[0534] These analyses strongly suggest that it is possible to
dissociate the beneficial cognitive effects of the methylthioninium
moiety of MTC from its undesirable haematological effects by
optimising the formulation. As discussed in a prior-filed
unpublished patent application (PCT/GB2007/001103, the contents of
which are herein specifically incorporated by reference), a novel
stabilised reduced salt form (designated "L-MTx") would have the
benefit of bypassing the reductase activity which is necessary for
absorption of the methylthioninium moiety of MTC. The stable L-MTx
has been found to have higher solubility than MTC, and upon
dissolution remains substantially in the uncoloured reduced state
for more than 1 hr, permitting direct absorption as the reduced
methylthioninium species. A further benefit of the stabilised L-MTx
may be that even higher efficacy could be achieved because higher
doses of the therapeutically active form could be absorbed without
limitation by the capacity of the gastric thiazine dye reductase
activity on the one hand, and haematological side effects and
diarrhea on the other. These are discussed further below.
[0535] As predicted from the present analysis, the L-MT salt form
has been found to have significantly less haematological toxicity
than MTC. FIG. 9 shows the differences between MTC and L-MTx across
a range of oral doses in terms of key red cell parameters in rats
dosed daily for 14 days. As can be seen, L-MTx-dosed animals had
higher counts of red cells ("RBC"), higher levels of haemoglobin
("HB") and higher red-cell haemoglobin concentration ("MCHC"). The
mean red-cell volume was less ("MCV"), indicating that more mature
red cells were released from the bone marrow, and the
reticulocytosis induced by the haemolytic effects of MTC was
reduced ("RETI").
TABLE-US-00004 TABLE 4 Statistical analysis of differences in key
red cell parameters in rats between MTC and L-MTx doses. Dose
(mg/kg) Difference with respect to MTC p-value Haemoglobin (g/dL)
0.sup.(1) -0.39 0.427 15.sup.(2) 0.80 0.106 45.sup.(2) 1.43 0.00465
150.sup.(2) 3.03 <0.0001 Mean cell haemoglobin concentration
(g/dL) 0.sup.(1) 0.26 0.780 15.sup.(2) 0.80 0.392 45.sup.(2) 1.93
0.0414 150.sup.(2) 6.05 <0.0001 Mean cell volume (fL) 0.sup.(1)
0.08 0.961 15.sup.(2) -1.18 0.475 45.sup.(2) -7.07 <0.0001
150.sup.(2) -9.14 <0.0001 Red cell count (10.sup.6/mL) 0.sup.(1)
-0.27 0.171 15.sup.(2) 0.41 0.041 45.sup.(2) 1.17 <0.0001
150.sup.(2) 1.06 <0.0001 Reticulocytes (% of red cells)
0.sup.(1) -0.08 0.973 15.sup.(2) -0.54 0.816 45.sup.(2) -6.53
0.0063 150.sup.(2) -7.59 0.0022 .sup.(1)The p-value is from a test
of whether the value of the vehicle-only dose is significantly
different from zero. .sup.(2)The p-value is from a test of whether
the value is significantly different from the vehicle-only
dose.
Example 5
Available Studies
[0536] As can be seen from the foregoing discussion, the
optimisation of an appropriate therapeutic dose of MTC and its
formulation are complex. A major barrier to this is the lack of a
suitable pharmacokinetic model. Although there have been attempts
to generate a PK model, these are contradictory and do not take
account of all of the available data. Therefore, a completely novel
approach to development of a PK model was required. Before
presenting this, the available data and models are summarised.
[0537] There are 3 published studies of MTC in humans. These are
first summarised, and then discussed together. There is a further
published study in humans (Rengelshausen et al., (2004)
Pharmacokokinetic interaction of chloroquin and methylene blue
combination against malaria. Eur. J. Clin. Pharmacol. 60: 709-715)
which is not used further in the present document, as its
methodology and findings are similar to those of Peter et al.
(2000) discussed below.
1) Prior Art Study 1
[0538] The first systematic reference studies were carried out by
DiSanto and Wagner (1972) and reported in a series of three papers,
two of which are summarised below.
[0539] 1a) DiSanto A R and Wagner J G (1972a) Pharmacokinetics of
highly ionized drugs I: whole blood, urine and tissue assays. J
Pharmaceut Sc 61: 598-601
[0540] The paper reports a method for analysis of MTC in whole
blood, urine and tissues. In essence, the method consists in
preparing the aqueous matrix with a high salt concentration
(>2M), extracting MTC into dichloroethane, and measuring
absorbance of the total dichlororethane extract at 660 nm. A
stabilised leuco-form of MTC ("leuco-MTC") was found in urine, but
not identified chemically. This could be analysed by first
converting it to "free-MTC" by adding 5 N HCl and heating in a
boiling water bath for 2 min prior to extraction into
dichloroethane. The difference between the MTC recovered from urine
following acid treatment and MTC recovered without acid treatment
("free-MTC") was reported as "leuco-MTC".
[0541] 1b) DiSanto A R and Wagner J G (1972b) Pharmacokinectis of
highly ionized drugs II: absorbtion, metabolism and excrection in
man and dog after oral administration. J Pharmaceut Sc 61:
1086-1090.
[0542] In this study, 7 adult male volunteers aged between 21 and
40 years and weighing between 54.5 and 95.3 kg ingested 10 mg of
MTC USP. Urine was collected in the intervals tabulated below.
Average urinary excretion rates for oxidised-MT ("Ox-MT", also
referred to as "free-MB") and leuco-MT ("L-MT") with corresponding
standard errors are shown in Table 5, and in FIGS. 10 and 11.
TABLE-US-00005 TABLE 5 Excretion rates and standard error ("se")
for oxidised MTC ("Ox-MT") and reduced MTC ("L-MT") from DiSanto
and Wagner (1972). Ox-MT Time (hr) Mid-time (hr) (.mu.g/hr) se-Ox
L-MT (.mu.g/hr) se-L 0.5 0.25 2.31 1.06 14.01 6.98 1 0.75 20.59
5.05 385.06 97.98 2 1.5 38.66 7.50 659.14 104.79 3 2.5 50.56 14.50
474.29 96.93 4 3.5 40.66 8.76 384.43 49.37 6 5 53.01 12.37 290.50
49.26 9 7.5 42.86 17.55 120.29 29.91 24 16.5 37.99 6.43 78.72 13.77
33 28.5 24.34 7.53 41.87 9.91 48 40.5 11.02 2.43 26.77 5.18 57 52.5
5.00 1.16 14.11 5.49 72 64.5 4.98 1.31 7.88 2.29 81 76.5 2.53 0.69
6.39 2.05 96 88.5 1.74 0.48 3.09 1.53 105 100.5 1.23 0.43 3.02 1.60
120 112.5 0.88 0.28 1.91 0.83
TABLE-US-00006 TABLE 6 Urinary excretion data for Ox-MTC and L-MT
Parameter Free Leuco Kel 0.2263 0.2430 K12 0.7506 0.2962 K21 0.2381
0.1040 Ka 0.1626 0.9654 Tlag (hr) 0.2078 0.2381 VcF (L) 29.7918
8.2607 Correlation (means, obs vs pred) 0.9878 0.9920
Non-compartmental secondary parameters F 0.1483 0.4982 Vc (L)
4.4188 4.1152 Cl (L/hr) 6.7420 2.0074 AUC (.mu.g hr) 1483.24
4981.55 Urinary excretion (% of total) 22.94% 77.06% MRT (hr)
24.5000 16.8774 T1/2 (distribution, hr) 0.5930 1.1530 T1/2
(elimination, hr) 15.0364 16.4953
[0543] The following standard abbreviations are used in the table:
Kel (terminal elimination rate constant), K12 (rate constant for
transfer from putative compartment 1 to compartment 2), K21 (rate
constant for transfer from putative compartment 2 to compartment
1), Ka (absorption rate constant, Tlag (absorption time-lag before
drug appears in central (ie blood) compartment). VcF (Vc.times.F),
F (calculated bioavailability), Vc (theoretical volume of
distribution of the drug in the central compartment), AUC (area
under the curve, a measure of total drug in blood), MRT (mean
residence time, time for 63.2% of administered dose to be
eliminated), T 1/2 (half-life).
[0544] From the urinary excretion data, Ox-MT and L-MT differ with
respect to distribution phase and apparent bioavailability.
However, the terminal elimination half-life (.about.16 hr) and
corrected apparent central volume (4 L) are comparable (Table 6).
Total urinary recovery is 6.465 mg (i.e. 65% of dose), of which 23%
is excreted as Ox-MT and 77% is excreted as L-MT.
2) Prior Art Study 2
[0545] This is described in Peter C, Hongwan D, Kupfer A,
Lauterberg B H (2000) Pharmacokinetics and organ distribution of
intravenous and oral methylene blue. Eur J Clin Pharmacol 56:
247-250.
[0546] In this study 7 human volunteers (4 males, 3 females) aged
19-53 were given MTC 100 mg (313 .mu.M) on 3 occasions at least 1
week apart as either a single IV injection (20 mg/ml in 0.9% NaCl
over 30 sec) or two 50 mg capsules in gelatine, or two 50 mg
capsules in gelatine together with 800 mg of Mesna (sodium
mercaptoethanesulphonate). The pharmacokinetic effect of
co-administration of Mesna was included because of the clinical use
of MTC in cancer chemotherapy regimes based on ifosfamide for which
Mesna is co-administered to prevent urotoxicity.
[0547] The analytical methodology for blood differed from that used
by DiSanto and Wagner in the following respects: [0548] Inclusion
of an internal standard [0549] Use of sodium hexanesulphonate as an
ion-pair to enhance extraction into dichloroethane [0550]
Chromatographic separation using a Nucleosil 100-5 CN column with
an isocratic mobile phase, with efflux monitored at 660 nm.
[0551] Peter et al. also measured urinary excretion of Ox-MT and
L-MTC to 24 hr, but reported only means of total excretion at
intervals ending at 2, 4, 6, 10, 14, 24 hr post-dose. The
analytical method in urine was said to be essentially identical to
that of DiSanto and Wagner.
[0552] The results are not tabulated by the authors, but are shown
graphically as reproduced in FIGS. 14 and 15.
[0553] The data have been read from these graphs and are tabulated
below.
TABLE-US-00007 TABLE 7 Concentration of Ox-MT in whole blood after
IV administration of 100 mg of MTC. Time Blood Ox-MT (hr)
(.mu.mol/L) 0.09 6.06 0.15 3.32 0.24 1.73 0.33 1.65 0.5 0.78 0.65
0.61 0.83 0.39 1.01 0.41 1.99 0.26 4 0.18
TABLE-US-00008 TABLE 8 Concentration of Ox-MT in whole blood after
oral administration of 100 mg MTC (mean of with and without Mesna).
Time Blood Ox-MT (hr) (.mu.mol/L) 0 0 0.09 0.00064 0.15 0.0011 0.24
0.0064 0.33 0.017 0.5 0.041 0.83 0.055 1.01 0.064 1.99 0.069 4
0.038
[0554] Peter et al report the following pharmacokinetic parameters
(Table 9).
TABLE-US-00009 TABLE 9 Pharmacokinetic parameters reported by Peter
et al. (2000) for MTC administered by intravenous and oral routes.
Parameter IV Oral AUC (.mu.mol/min/ml) 0.134 0.011 Cl (L/hr).sup.1
3 % of dose excreted in urine 28.6 18.6 at 24 hr Estimated
elimination T1/2: blood (1-4 hr, hr) 5.25 urine (4-24 hr, hr) 6.6
.sup.(1)Cl: clearance, the volume of blood cleared of drug in unit
time.
[0555] Peter et al. further note that the fraction of total MT
excreted in the urine in the L-MB form was approximately 1/3 of the
total, and this did not differ between oral and IV dosing.
3) Prior Art Study 3
[0556] This is described in Moody J P, Allan S M, Smith A H W,
Naylor G J (1989) Methylene blue excretion in depression. Biol
Psychiat 26: 847-858.
[0557] This is a limited study of 24-hr urinary excretion during a
3-week trial period in depressed subjects taking 15 mg/day (5 mg
t.i.d.) or 300 mg/day (100 mg t.i.d.). Twenty-four hr urine
collections were obtained in 7 subjects at the end of 7, 14 or 21
days treatment. The analytical method was said to be that of
DiSanto and Wagner. The results are summarised below in Table
10.
TABLE-US-00010 TABLE 10 Summary of data on urinary excretion of MTC
in humans from the study by Moody et al. (1989). Ox-MT Total-MT
(mg) L-MT (mg) (mg) Repeat Dose Study (15 mg/24 hr) Days 7 6.1 7.2
14 5.3 8 21 6.1 6.4 24 hr urinary excretion (mg) 5.8 7.2 % of total
urinary excretion 44.8% 55.3% F (apparent bioavailability) 0.39
0.48 Repeat Dose Study (300 mg/24 hr) Days 7 43.9 75.6 14 41.1 71.6
21 45.2 60.4 24 hr urinary excretion (mg) 43.4 69.2 % of total
urinary excretion 38.6% 61.5% F (apparent bioavailability) 0.14
0.23 Single Dose Study Dose mg) 25 14.9 2.8 17.7 50 28.1 2.7 30.8
100 33.5 6 39.5 % of total urinary excretion 25 84.2% 15.8% 50
91.2% 8.8% 100 84.8% 15.2% F (apparent bioavailability) 25 0.60
0.11 0.71 50 0.56 0.054 0.62 100 0.34 0.060 0.40
[0558] The single-dose data from this study has been combined with
that of the DiSanto & Wagner and Peter et al. studies to
provide an estimate of apparent oral bioavailability based on
urinary excretion at 48 hr of total-MT.
Discussion of Key Results
[0559] There are several respects in which the models developed on
the basis of the data tabulated above are inconsistent. The most
important is that the terminal elimination half-life deduced by
Peter et al. (5.5-6.3 hr) from analysis of blood concentration data
is inconsistent with the terminal elimination half-life deduced by
DiSanto and Wagner (15-16.5 hr) from urinary excretion data. It is
also inconsistent with long discolouration of urine observed
following intra-operative IV administration of MTC to localise
parathyroid glands for surgery (Kuriloff and Sanborn, 2004). The
problem arises because Peter et al. (2000) have based their
estimates on blood data obtained of 4 hr, or 12 hr in the case of
Rengelshausen et al. (2004) who followed the same pharmacokinetic
approach. These analyses fail to take account of the terminal
elimination phase, because of technical difficulties encountered in
estimating Ox-MT levels in blood, even using LC-MS (Liquid
Chromatography--Mass Spectroscopy) after the blood levels fall
below detection limits. The terminal elimination phase can be
better analysed using urinary excretion data. Although it is well
known that the urinary excretion rate can provide a valid way of
estimating the elimination rate constant in simple systems (eg
Gibaldi and Perrier (1982) Pharmacokinetics), the problem with the
available MTC data is that is complex, and there is no obvious way
to link the blood data and urinary excretion data into a single
coherent integrated model able to account both for the IV and oral
dosing cases. Providing a solution to this problem is crucial for
the development of a suitable predictive model which can be used to
optimise dosing of MTC or other MT forms for the treatment of AD
and in other therapeutic contexts. The solution to this problem is
discussed below.
Example 6
Development of Integrated Pharmacokinetic Model
i) Oral Bioavailability
[0560] The Peter et al. data provide a useful indication of blood
levels following oral vs IV administration. Comparison of the AUC
values over the 4 hr time-period indicates that blood levels
following oral administration are 8.2% of those seen after IV
administration. However, this estimate cannot be used to determine
oral bioavailability. It is inconsistent with the Peter et al.
urine recovery data at 24 hours, where urinary recovery following
oral dosing was found to 65% of that obtained after IV dosing (see
Table 5). This figure is comparable with the urinary excretion data
obtained from the DiSanto and Wagner and the Moody et al.
studies.
[0561] The data from these studies are combined in FIG. 16 to
provide an overall estimate of oral bioavailability. It suggests a
figure between 40%-80% depending on dose over the range 10-100 mg.
It is also apparent from FIG. 16 that there is dose-dependent
reduction in bioavailability as determined by urinary recovery
following oral dosing.
[0562] There is therefore a discrepancy between the estimate of
oral bioavailability determined from direct measurement in blood
and that determined from urinary excretion. This implies that the
low blood levels seen in blood following oral dosing cannot be
explained simply by a limitation in absorption as suggested by
Peter et al. (2000). The low blood levels seen after oral
administration are more likely to reflect a difference in the
apparent volume of distribution for MTC administered orally and by
the IV route. Rapid early tissue uptake was confirmed by DiSanto
and Wagner who reported that 29.8% of the intravenous dose of MTC
could be recovered in heart, lung, liver and kidney at 2 minutes
following administration in rat. This picture of an early rapid
distribution phase followed after 10 hrs by a slow elimination
phase is also consistent with the urinary excretion data shown in
FIGS. 12 and 13. Therefore, blood data collected over a 4 hr time
course as provided by Peter et al. are not sufficient to derive a
valid estimate of redistribution of MT between absorption, central
and peripheral compartments.
ii) Model Constructed by Combining Blood Data from Peter et al.
(Tables 7&8) and Urinary Excretion Data from DiSanto and Wagner
(Table 5)
[0563] One approach to deriving a pharmacokinetic model from the
available studies is to use linear differential equations to
determine directly a system of compartments which can be fitted to
the available data sets. The data used are the DiSanto and Wagner
urinary excretion data set for 7 subjects listed in Table 5, taking
account of differential excretion of Ox-MT and L-MT. This is
combined with the Peter et al blood level data listed in Tables 7
and 8, also based on 7 subjects. It is assumed that the DiSanto and
Wagner data can be linked to both IV and oral blood concentration
data sets after appropriate scaling on the basis that the urinary
excretion profiles as determined by Peter et al. were similar for
the 2 routes of administration (FIG. 17).
[0564] However, the DiSanto and Wagner urinary excretion data set
is used for fitting in preference to the Peter et al. data because
the latter does not explicitly take account of differential
excretion of Ox-MT and L-MT, and because the sampling intervals are
coarse relative to those available from the DiSanto and Wagner data
set.
[0565] The modelling was done in two stages:
[0566] In the first stage the Peter et al. blood concentration data
to 4 hr following a single IV dose of 100 mg of MTC was combined
with the DiSanto and Wagner urinary excretion data set to 120 hr
for single oral MTC dose of 10 mg. The second stage was to see if
the same or similar compartment system can be used to fit the Peter
et al. blood concentration data following a single oral dose of 100
mg of MtC, combined with the DiSanto and Wagner urinary excretion
data set to 120 hr for single oral MTC dose of 10 mg. In both
cases, scaling parameters to allow for the 10-fold difference in
dose were estimated by the corresponding models.
[0567] FIG. 18 shows the best distribution of compartments and
corresponding rate constants which could be fitted to the three
data sets (Peter et al IV-dosing blood concentration data [Table
7], DiSanto and Wagner urinary Ox-MT data [Table 5] and urinary
L-MT data [Table 5]). The central compartment is C2. The scaling
parameters to allow for the fact that there was a 10-fold
difference in the doses used in the blood and urine data sets were
explicitly estimated by the model for urinary Ox-MT (S-Ox) and
urinary L-MT (S-L) and are shown in Table 11. The solution to the
model requires two peripheral compartments, shown as C3 and C4 in
FIG. 18, and a further excretion compartment (C5). There are two
outputs from C5, one which represents scaled observed urinary
excretion of L-MT (designated K50 in Table 11), and a second output
which represents an unmeasured loss (designated K500 in Table 11),
which is presumed to represent secondary hepatic metabolism of MT
which is excreted through the bile as an unmeasured metabolite. The
output from C3 (designated K30 in Table 11) represents the quantity
measured as urinary Ox-MT. The percentages shown represent
partitions of predicted total excretion at 120 hr, estimated from
the corresponding AUC values.
[0568] The parameters estimated by the model are listed below in
Table 11.
TABLE-US-00011 TABLE 11 Model parameters estimated for a single
intravenous dose of 100 mg MTC. The rate constants are as indicated
in FIG. 18. K50 is the urinary excretion rate constant from C5, and
K500 is the presumptive hepatic excretion rate constant from C5. V2
is the apparent volume of distribution of MT in C2 calculated by
the model. S-Ox and S-L are the scaling parameters calculated by
the model to account for the fact that urinary data came from an
experiment in which MTC was administered as a 10 mg oral dose, and
the blood data came from an experiment in which MTC was
administered as a single 100 mg IV dose. Parameter Estimate K23
1.60 K24 3.94 K30 0.0093 K32 0.088 K42 0.87 K45 0.28 K50 0.78 K500
0.081 S-Ox 10.6 S-L 6.3 V2 66.03 Correlations (observed vs
predicted): Blood 0.98 Urinary Ox-MT 0.96 Urinary L-MT 0.98
[0569] In the second stage, the same basic model was fitted to the
Peter et al. blood concentration data following a single oral dose
of 100 mg of MTC (Table 8), and scaled urinary excretion data from
DiSanto and Wagner (Table 5) following a single oral dose of 10 mg
of MTC.
[0570] FIG. 22 shows the best distribution of compartments and
corresponding rate constants which could be fitted to the three
data sets (Peter et al oral-dosing blood concentration data [Table
7], DiSanto and Wagner urinary Ox-MT data [Table 5] and urinary
L-MT data [Table 5]). The oral model assumes two further
compartments prior to the central compartment (C2). These are C1
(the primary absorption compartment, assumed to correspond to
stomach), and a second pre-central compartment (C6, presumed to
represent a first-pass metabolism hepatic compartment). There is a
loss from C1 (designated K100 in Table 12) which is presumed to
represent non-absorbed MTC, and further loss from C6 (designated
K600 in Table 12) which is presumed to represent loss due to first
pass metabolism. The scaling parameters to allow for the fact that
there was a 10-fold difference in the doses used in the blood and
urine data sets were explicitly estimated by the model for urinary
Ox-MT (S-Ox) and urinary L-MT (S-L) and are shown in Table 12. As
for the IV model, the solution to the model requires two peripheral
compartments, shown as C3 and C4 in FIG. 22, and a further
excretion compartment (C5). There are two outputs from C5, one
which represents scaled observed urinary excretion of L-MT
(designated K50 in Table 12), and a second output which represents
an unmeasured loss (designated K500 in Table 12), which is assumed
to represent secondary hepatic metabolism of MT which is excreted
through the bile as an unmeasured metabolite. The output from C3
(designated K30 in Table 12) represents the quantity, measured as
urinary Ox-MT. The percentages shown represent partitions of
predicted total output from the system excretion at 120 hr,
estimated from the corresponding AUC values.
[0571] The parameters estimated by the model are listed below in
Table 12.
TABLE-US-00012 TABLE 12 Model parameters estimated for single oral
dose of 100 mg MTC. Parameter 1 Estimate K100 0.44 K16 1.68 K23
1.39 K24 0.67 K30 0.016 K32 0.091 K42 0.00095 K45 2.059 K50 1.45
K500 0.61 K600 0.20 K62 0.35 S-Ox 12.3 S-L 19.6 Vc2 (L) 319.9
Correlations (observed vs predicted): Blood Ox-MT 0.99 Urinary
Ox-MT 0.98 Urinary L-MT 0.99
[0572] Scaling factors for urinary Ox-MT and L-MT from DiSanto and
Wagner (10 mg dose, oral) to fit with Peter et al data (100 mg
dose, oral) are explicitly estimated for the oral version of the
model as S-Ox and S-L respectively. A further modification required
to achieve a fit for the oral data was the introduction of a time
delay for the urinary excretion data from DiSanto and Wagner. This
delay was estimated as a non-linear function ranging from 0.2 to 1
hr for excretion times earlier than 1 hr, and a constant time delay
of 1 hr thereafter.
[0573] As can be seen in Table 11 and 12, there were very high
correlations (all greater than 0.96) between the model outputs and
the input data sets, as can also be readily seen from FIGS. 19-21
and 23-25. The model therefore provides a close fit to the
experimental data.
iii) Comparisons of Model Outputs with Other Data Sources
[0574] As a check of the oral model, its outputs were compared with
other available data sets. The outputs of the oral model (FIG. 22)
were first compared with the urinary excretion rates reported by
Peter et al. (2000) and shown above in FIG. 18. This comparison is
shown below in FIG. 26. There was good overall agreement, apart
from the 2-4 hr collection interval, when the level reported by
Peter et al. was half of that predicted by the model. Excluding
this value, the correlation between the two was 0.86. The total
24-hour excretion predicted by the model and that reported by Peter
et al. is also compared in FIG. 26. The model predicts that total
urinary excretion was 23% of the dose, whereas the Peter et al.
estimate was 18.6%.
[0575] As a further check on the model, the total predicted 48-hour
urinary excretion was compared with the data shown above in FIG.
16, which compiles the urinary excretion data from DiSanto and
Wagner and Moody et al. This is shown again in FIG. 27, with the
model output indicated by "M", and the Peter et al. data indicated
by "P".
[0576] Finally, a comparison was made between the compartment
predictions and the results from an oral study in which pigs were
administered a single 20 mg/kg dose, and brain levels of MT were
determined. Pigs were given a single oral administration of MTC at
a target dose level of 20 mg/kg bodyweight. Blood (0.5, 1, 2, 4, 8,
12, 24 and 48 h) and urine (1, 2, 3, 4, 5, 6, 7, 8, 12, and 24 h)
were collected at regular timepoints up to 48 hrs. Two animals were
sacrificed at each of 1,8, 24 and 48 h post dose and brain samples
retained. Pharmacokinetic evaluation of the free base of MTC was
performed on whole blood and brain tissue samples. Two batches of
brain tissue sample were extracted for each animal and analysed
essentially as described by Peter et al (2000).
[0577] Brain tissue (500 mg) was vortexed and then extracted with
dichloroethane (5 ml) and the organic phase taken to dryness under
nitrogen. The extract was taken up with methanol and separated by
reverse-phase HPLC with ultraviolet detection. The method was
validated, using internal standards, over the range of 10 to 2000
ng of MTC per gram of tissue. The mean inter-occasion accuracy for
MTC was 107%, 95% and 105% at 20, 100 and 1600 ng/g, respectively
and the coefficient of variation at each level, was not more than
20%.
[0578] The terminal elimination half-life in the pig was found to
be 23.5 hours for both blood and brain, consistent with the urinary
excretion findings of DiSanto and Wagner indicating that the
terminal elimination phase is much longer than estimated either by
Peter et al. (2000) or Rengelshausen et al. (2004).
[0579] In order to use the pig data to determine which human model
compartment predicts the brain levels, the time-base for the pig
data was rescaled to correspond to the human half-life (15.7
hr).
[0580] The results are shown in FIG. 28. All compartments have been
rescaled to their respective maxima. It can be seen from FIG. 28
that the central compartment (C2, blood) and C4 follow each other
very closely, indicating that MT is freely exchangeable between C2
and C4.
[0581] On the other hand, elimination of MT from the pig brain can
be seen to parallel the predicted elimination from C3, and not from
C4. Therefore, of the two inner compartments of the model (C4 and
C3), it can be seen that C3 provides a prediction of expected brain
levels.
iii) Interpretation of the Integrated Pharmacokinetic Model for
MTC
[0582] The main kinetic features of the IV and oral models are now
compared.
a) IV Human Model
[0583] The key kinetic features of the human intravenous model is
summarised in Table 13. The data have been normalised to the case
of a single 100 mg dose (313 .mu.M).
TABLE-US-00013 TABLE 13 Summary of the key kinetic features of the
human intravenous PK model. Intravenous model AUC- % AUC-
A-T1/2.sup.1 D-T1/2.sup.1 E-T1/2.sup.1 AUC.sup.2 out.sup.3
out.sup.3 Tmax.sup.4 MRT.sup.5 Central compartments C2 0.1 1.4 17.9
286 16.3 C4 0.1 1.4 17.9 984 0.5 17.1 Deep compartment C3 1.3 1.4
17.9 4645 4.0 26.6 Excretion compartment C5 0.6 1.6 17.9 320 2.0
18.2 Post-central outputs C500 2377 8.2% Ur-Ox-MT 3707 12.8%
Ur-L-MT 22836 79.0% Total outputs 28920 .sup.1For each of the
compartments, half-lives for an absorption-phase (A-T1/2), a
distribution-phase (D-T1/2) and an elimination phase (E-T1/2) have
been calculated in hr, using a tri-exponential approximation to the
model output data. .sup.2The AUC.sub..infin. (.mu.mol-hr/l) has
been calculated for MT in each of the "interior" compartments.
.sup.3The AUC.sub..infin. (.mu.mol-hr/l) has been calculated for MT
in each of the post-central compartments, and these have been shown
by percentage. .sup.4The Tmax is the calculated time (hr) after
dose at which the MT level in each interior compartment is maximum.
.sup.5MRT is the mean residence time in each compartment,
calculated as the time required for 63.2% of the administered dose
to be eliminated.
[0584] Central Compartments. As can be seen from Table 13, and also
from FIG. 28, the kinetic properties of MT in the C2 and C4
compartments are essentially identical, supporting the concept that
the form of MT in C4 is in ready exchange equilibrium between the
form measured as the blood level of Ox-MT in blood in C2. As the C4
compartment is the principal determinant of urinary excretion of
the L-MT form measured in urine, it is concluded that the C4 form
of MT represents the L-MT side of the L-MT-Ox-MT equilibrium which
exists in the body. After IV administration, the amount of MT in C4
reaches its maximum level within 30 minutes, and is thereafter
eliminated at the common terminal elimination rate.
[0585] Deep Compartment. By contrast, it can be seen that C3 in the
IV case has different dynamic properties. It takes 4 hr after
administration for the maximum C3 level to be reached, and the mean
residence time in C3 is substantially longer than in either C2 or
C4. In light of the pig brain data, it is inferred that the C3
compartment represents the pool of MT which is kinetically trapped
inside cells as described by May et al. (2004).
[0586] According to May et al. (2004), MT needs to be in the L-MT
form in order to cross the cell membrane. Inside the cell, there is
a new L-MT-Ox-MT equilibrium which is determined by a combination
of the predominant reducing environment in the intracellular
milieu, and the prevailing pH inside the cell (.about.pH 7).
Experiments in vitro (not shown) have indicated that it is very
difficult to keep MT in the reduced state at pH 7 using
physiologically acceptable reducing agents at physiologically
acceptable concentrations. That is, at pH 7, MT would tend to exist
predominantly in the Ox-MT state were it not for the predominantly
reducing conditions which are maintained within the cell. However,
in the Ox-MT form, MT cannot diffuse out of the cell. This creates
conditions for a new equilibrium whereby MT is trapped within
cells, leading to accumulation of intracellular MT against a
concentration gradient, which can be demonstrated in tissue culture
(not shown). This explains the otherwise paradoxical
pharmacokinetic observation that MT is both rapidly distributed to
tissues following IV administration (as reported by DiSanto and
Wagner), but nevertheless eliminated much more slowly. Thus DiSanto
and Wagner found that within 2 minutes of a dose administered IV in
rats, approximately 25% could be recovered from the major
organs.
[0587] According to the human IV model, the level of MT which is
measured in urine as the Ox-MT form is closely related kinetically
to the species which is trapped within an intracellular
environment, including the brain, as indicated by the pig brain
data.
b) Oral Human Model
[0588] The key kinetic features of the human oral model is
summarised in Table 14. The data have been normalised to the case
of a single 100 mg dose (313 .mu.M).
TABLE-US-00014 TABLE 14 Summary of the key kinetic features of the
human oral PK model (for details see footnotes to Table 13). Oral
Model AUC- % AUC- A-T1/2 D-T1/2 E-T1/2 AUC out out Tmax MRT Input
compartments C1 154 0.5 C6 467 0.9 2.4 Central compartments C2 0.5
1.3 15.7 184 2.0 15.7 C4 0.5 1.3 15.7 60 2.0 16.0 Deep compartment
C3 1.4 1.3 15.7 2345 5.0 25.0 Excretion compartment C5 0.7 1.4 15.7
61 2.0 16.5 Pre-central outputs C100 7329 23.0% C600 9869 31.0%
Pre-central outputs C500 3422 10.7% Ur-Ox-MT 3202 10.0% Ur-L-MT
8060 25.3% Total outputs 31882
[0589] Primary absorption compartment. In the oral model, there are
2 input compartments (C1 & C6) prior to the appearance of MT in
the central compartments (C2 & C4). As discussed above in the
section Relationship Between Cognitive and Haematological Activity,
the properties of C1 are crucial in determining the bioavailability
and form in which MT is absorbed. As shown in Table 14, the mean
residence time in C1 is 30 minutes. It can be calculated that 50%
of MT has been absorbed by 30 minutes, and that 90% of MT has been
absorbed from C1 by 1 hr. This indicates that C1 is the stomach,
where the low pH (pH .about.2) favours the enzyme-mediated
conversion of MT to the L-MT form which is readily absorbed (May et
al., 2004). It is important to note that 23% of administered MTC
escapes absorption, and is thereafter lost (shown as C100 in Table
14). Therefore, absorption from C1 is also critical in determining
how much MTC passes through the gastro-intestinal tract to the
distal gut where the mild antibiotic activity of MTC causes
diarrhea by repopulation of distal gut flora. The properties of C1
are therefore crucial for optimising the absorption and efficacy of
MTC, and minimising the side effects, both from unabsorbed MTC
(diarrhea) and from late-absorbed MTC as shown in the clinical
trial (haematological side effects).
[0590] Central Compartments. As for the IV model, the kinetic
properties of the C2 and C4 compartments are essentially identical
in the oral model. The significant difference between the IV and
oral cases is that the rate constant K24 (3.94) is very is 4.times.
higher in the IV case than in the oral case (K24: 0.67). This
indicates that following IV administration, there is a major flux
of the administered Ox-MT to the L-MT form. By contrast, in the
oral case, the bulk of MT has already been reduced to the L-MT form
prior to entry into the central compartments.
[0591] A further significant difference which can be seen between
the IV model and the oral model is that the estimated apparent
volume of distribution of MT is very much greater in the oral case
(320 L, Table 12) than in the IV case (66L, Table 11). This almost
4-fold difference is the main explanation for the low concentration
of Ox-MT observed in the blood following oral administration than
after IV administration. This was explained erroneously by Peter et
al. (2000) as a low bioavailability. Although it is true that
approximately half the orally administered dose is lost by a
combination of non-absorption (the C100 loss in Table 14 and FIG.
22), and first-pass metabolism (the C600 loss in Table 14 and FIG.
22), the C2 AUC in the oral case is 64% of the C2 AUC in the IV
case. Therefore the apparent bioavailability as determined by blood
AUC ratios is very close to the apparent bioavailability calculated
from the DiSanto and Wagner urine excretion data, which indicated
that 65% of the administered dose could be recovered in urine for
the 10 mg dose case.
[0592] Deep Compartment. The maximum level of MT is seen in the
central compartments 2 hr after administration. By contrast, the
peak level is reached in the deep compartment (C3) only at 5 hr
after administration. Again the mean residence time of MT in C3 is
much longer than in the central compartments (25 hr vs 16 hr).
Therefore, the features of C3 are essentially identical in the IV
and oral dosing models.
[0593] It is important to compare the apparent bioavailability of
MT in C3, which is representative of brain levels, between the oral
and IV dosing routes. The oral C3 AUC is 50% of the IV C3 AUC.
Therefore essentially half of the oral dose is available within
cells compared to the IV case.
Example 7
Dosing Implications of Integrated Pharmacokinetic Model
[0594] An integrated pharmacokinetic model is a critical tool
required for: [0595] optimisation of dosing regime [0596]
optimisation of formulation [0597] establishing relationship
between blood-level and efficacy
[0598] The key planning parameter that can be derived from the
pharmacokinetic model is the prediction of steady-state levels
achieved on repeated dosing. It will be evident that a model which
assumes a terminal elimination half-life of 5-6 hr (Peter et al.,
2000; Rengelshausen et al., 2004) will produce quite different
estimates of the optimal dosing regime to one in which the
elimination half-life is 16 hr. It can be estimated from the
integrated model which has been developed that a dosing regime of
3/day will have quite different implications as regards predicted
steady-state levels assuming an elimination half-life of 6 hr vs 16
hr. Thus, if the Peter et al. estimate were true, then the
accumulation factor (R, ie the ratio of steady state level to
single dose level) that would be seen for 8-hourly dosing would be
1.4. By contrast, if the estimate of 16 hr is true, then the
corresponding value of R is 4.8 for 8-hourly dosing. This implies
that there would be a 3.4-fold difference in the expected
steady-state level of MT (in blood and in brain) according to the
two models. It is therefore difficult to determine an accurate
relationship between dose and efficacy or side effects without a
valid pharmacokinetic model.
[0599] The key intervening variable linking dose and efficacy is an
estimate of the steady-state levels of MT in critical compartments
at varying regular dosing frequencies. The model permits these to
be determined as the predicted average steady state levels in C2
and C3, as shown in Table 15.
TABLE-US-00015 TABLE 15 Predicted mean steady-state levels of MT in
compartment C2 and C3 as a function of dosing frequency (values in
.mu.mol). C2 C3 3/day 4.8 295.5 2/day 3.2 197.0 1/day 1.6 98.5
Correlation Between Observed and Predicted Clinical Efficacy Based
on the Integrated Oral Human Pharmacokinetic Model
[0600] We first examine the relationship between the observed
clinical efficacy (effect size in ADAS-cog units at 50 weeks) and
the predicted steady-state level of MT in the deep compartment (C3)
which is, as discussed above, correlated with measured brain levels
in pig. That is, the quantity of MT in C3 and the concentration of
MT in brain are related by a constant which depends on the fraction
of MT which reaches the brain, and the accuracy of detection of
total MT in brain. As this scaling factor is at present unknown in
the human case, for the purpose of further discussion the quantity
of MT in C3 is taken as a proxy for the expected brain level. The
relationship is shown in FIG. 29.
[0601] As can be seen in FIG. 29, there is a very close
relationship between the predicted average steady state level of MT
in brain and the clinical effect size of Rember.TM. in TRx-014-001
for the 30 mg 3/day and the 60 mg 3/day doses. The relationship
does not hold for the 100 mg capsule for the reasons discussed
above in the section Relationship Between Cognitive and
Haematological Activity. In essence, the delay in dissolution of
the formulation of the 100 mg capsule used in TRx-014-001 did not
permit proportionate absorption of MTC in its therapeutically
active form.
[0602] An identical relationship can be defined between
steady-state blood level of MT (ie determined by C2) and effect
size, as shown in FIG. 30.
Dosing and Formulation Implications of Correlation Between Observed
and Predicted Clinical Efficacy Based on the Integrated Oral Human
Pharmacokinetic Model
[0603] From the foregoing analysis, there is the expectation of a
clear monotonic dose-response relationship between blood levels of
MT which can be measured clinically and effect size. From this,
appropriate nomograms can be calculated which take account of
measurement methodology. That is, efficacy could be related to
blood levels, and therapeutic blood levels could be specified using
appropriate analytical methodology.
[0604] A further implication of the relationship shown in FIG. 30
is to calculate the relationship between observed capsule
dissolution and the efficacy deficit, ie the difference in effect
size between observed effect size and predicted effect size. This
is shown in FIG. 31.
[0605] As can be seen from FIG. 31, there is a steep loss of
predicted efficacy as the observed percentage capsule dissolution
at 30 minutes drops below 20%. This confirms the conclusions
reached above in the section Relationship Between Cognitive and
Haematological Activity, and confirms that rapid dissolution is
critical for therapeutic activity. As discussed further in the
section Interpretation of the integrated pharmacokinetic model for
MTC, this can be explained by the critical role of the stomach in
the absorption of the MT moiety in its therapeutically active
form.
[0606] Therefore, in the design of an improved formulation of MTC,
the attainment of predicted efficacy is critically determined by
the requirement that the dissolution of the investigational
medicinal product (i.e. tablet or capsule) be greater than 50% in
30 minutes in standard conditions.
[0607] The relationships described herein have implications as
regards the conventional approach to achieving a more convenient
dosing regime, i.e. 2/day or 1/day. These dosing regimes would be
much more desirable in patients with dementia, who are forgetful
and hence need prompting to take medication. The conventional
approach to achieving a more convenient dosing regime is to create
a slow-release formulation. However, the present analysis indicates
that, on the contrary, a very high loading, slow-release
formulation of an MTC-based form of a therapeutic product would
essentially eliminate efficacy, as illustrated conveniently by the
properties of the 100 mg capsule in TRx-014-001.
[0608] A further inference which can be drawn from FIGS. 29 &
30 is that the dose of an MTC-based form of a therapeutic product
would need to be administered at a unit dosage of 120 mg or greater
to achieve a level of efficacy comparable to that seen in the
TRx-014-001 clinical trial with the unit dose of 60 mg administered
3 times per day.
[0609] A further inference which can be drawn from FIGS. 29 &
30 is that a unit dosage of 100 mg or more administered 3 times per
day would be required to achieve a level of efficacy higher than
that seen in the TRx-014-001 clinical trial. However, as discussed
in the section Summary of Phase 2 Clinical Trial TRx-014-001 there
is a limitation in the amount of MTC which can be administered in
the present formulation because of the increasing adverse
haematological effects and diarrhea at doses at or above 100 mg
3/day.
Implications for Improved Formulations and Dosing Regimes
[0610] As can be seen from FIG. 31A, there is a steep loss of
predicted efficacy as the observed percentage capsule dissolution
at 30 minutes drops below 20%.
[0611] Therefore, in the design of an improved formulation of MTC,
the attainment of predicted efficacy is critically determined by
the requirement that the dissolution of the investigational
medicinal product (i.e. tablet or capsule) be greater than 50% in
30 minutes in standard conditions.
[0612] The relationships described herein have implications as
regards the conventional approach to achieving a more convenient
dosing regime, ie 2/day or 1/day. These dosing regimes would be
much more desirable in patients with dementia, who are forgetful
and hence need prompting to take medication. The conventional
approach to achieving a more convenient dosing regime is to create
a slow-release formulation. However, the present analysis indicates
that, on the contrary, a slow-release formulation of an MTC-based
form of a therapeutic product would essentially eliminate efficacy,
as illustrated conveniently by the properties of the 100 mg capsule
in TRx-014-001.
[0613] A further inference which can be drawn from FIGS. 29 &
30 is that the dose of an MTC-based form of a therapeutic product
would need to be administered at a unit dosage of 120 mg or greater
to achieve a level of efficacy comparable to that seen in the
TRx-014-001 clinical trial with the unit dose of 60 mg administered
3 times per day.
[0614] A further inference which can be drawn from FIGS. 29 &
30 is that a unit dosage of 100 mg or more administered 3 times per
day would be required to achieve a level of efficacy higher than
that seen in the TRx-014-001 clinical trial. However, as discussed
in the section Summary of Phase 2 Clinical Trial TRx-014-001 there
is a limitation in the amount of MTC which can be administered in
the present formulation because of the increasing adverse
haematological effects and diarrhea at doses at or above 100 mg
3/day.
Correlation Between Observed and Predicted Haematological Side
Effects Based on the Integrated Oral Human Pharmacokinetic
Model
[0615] We now consider the relationship between the expected
steady-state level of MT in C2 (blood) and the haematological side
effects observed in the TRx-014-001 study. Loss of red cells at 24
weeks is taken as the most informative indicative variable. The
relationship is shown below in FIG. 31B.
[0616] As can be seen in FIG. 31B, the level of loss of red cells
is very much higher than the predicted steady state level of MT in
the blood. This is strongly confirmatory of the delayed dissolution
hypothesis outlined in the section Relationship Between Cognitive
and Haematological Activity. Specifically, according to the delayed
dissolution hypothesis, a quite distinct form of MT is responsible
for haematological side effects. This was postulated to be a dimer,
the formation of which is favoured in the alkaline conditions of
the small intestine and lower gut. Therefore, the haematological
side effects observed in TRx-014-001 were a specific consequence of
the gelatine capsule formulation used in the study, and are
unlikely to be an inherent feature of the MT moiety itself, if
absorbed via the stomach as described.
Example 8
Implications for Improved Compositions and Dosing Regimes
Absorption and Efficacy
[0617] As can be seen from the foregoing analysis, the limiting
factors in the level of therapeutic efficacy which could be
attained using an MTC-based medicinal product are a combination of
limitations in absorption and adverse effect limitations. The
present section discusses how these limitations could be overcome
in the light of the analysis made possible by the development of
the integrated pharmacokinetic model.
[0618] We first compare the actual dose with the effective dose in
FIG. 32, calculated using the same relationship discussed in FIG.
16.
[0619] As can be seen, the efficacy-limiting factor is a
combination of the limitation in absorption and first-pass
metabolism discussed above. These combine to limit severely the
benefit which could theoretically be achieved by increasing the
dose. Indeed the apparent efficacy plateau suggested above in FIG.
7 is determined almost entirely by the limitation in the effective
dose which can be delivered using a medicinal product based on the
present form of MTC.
[0620] Prior filed unpublished application PCT/GB2007/001103
describes certain stabilised reduced salt forms of the
methylthioninium moiety (referred to in what follows as "L-MTx").
We here use the integrated pharmacokinetic model, and the
relationship defined by it with therapeutic efficacy observed in
TRx-014-001 to determine how this novel composition of matter could
be used to optimise treatment of AD based on the methylthioninium
moiety.
[0621] We first consider the predicted fraction of orally
administered L-MTx that would be expected to be absorbed. This is
calculated on the basis that the loss due to first-pass metabolism
(ie the loss from C6 designated C600 in Table 14 and shown in FIG.
22) would not be eliminated by dosing with the L-MTx form. However,
it is expected that the loss due to initial non-absorption from C1
(ie the loss from C1 designated C100 in Table 14 and shown in FIG.
22) would be eliminated by dosing with the L-MTx form. This is
because the L-MTx form (particularly the dihydrobromide salt,
PCT/GB2007/001103) has more than twice the solubility of MTC, and
would be expected to bypass the thiazine-dye reductase (May et al.,
2004) which is presumed to exist in the stomach and is presumed to
be necessary for absorption. Based on these assumptions, the
predicted fraction of dose absorbed, calculated from the data
provided by the model is shown in FIG. 33. Specifically, the total
pre-central compartment losses amount to 54% of the administered
dose for the 100 mg case. Of this total loss, 43% is due to
non-absorption from C1. This is applied across doses to estimate
the expected bioavailibity of administered L-MTx allowing for loss
due to subsequent first-pass metabolism.
[0622] Once absorption into the central compartment has occurred,
the predicted efficacy can be determined from the relationships
described above linking steady state level in C3 or C2 with
observed effect size. These are shown for C3 in FIG. 34.
[0623] The corresponding relationships between expected clinical
efficacy of an L-MTx-based form of the methylthioninium moiety and
predicted average steady state level of MT in C2 (blood) for a
range of dosing regimes from 1/day to 3/day are shown in FIG.
35.
[0624] As can be seen from FIGS. 34 & 35, it is predicted that
a level of efficacy of -8.1 ADAS-cog units could be achieved on a
dosing regime of 100 mg of the L-MTx form administered twice daily,
which could also be achieved by dosing with 60 mg 3 times per day.
Even higher efficacy levels would be expected using 100 mg or
higher administered 3 times per day.
[0625] It is therefore inferred that substantially higher efficacy
and superior dosing regime could be achieved using the L-MTx form
of the methylthioninium moiety.
Safety and Tolerability of the L-MTx Form of the Methylthioninium
Moiety
[0626] As discussed above in the section Relationship Between
Cognitive and Haematological Activity, a significant limitation in
the extent to which higher doses of MTC could be administered to
achieve better efficacy is due to the combined consequences of
increasing haematolotical side effects and poor tolerability due to
diarrhea. Although it is likely that the L-MTx form would
substantially reduce diarrhea, it is not clear what the expected
haematological effects would be.
[0627] As shown in FIG. 9 and Table 4 in the section Relationship
Between Cognitive and Haematological Activity, it is expected that
the L-MTx form would have less haematological side effects based on
the rat studies discussed above. Furthermore, as discussed above in
the section Correlation between observed and predicted
haematological side effects based on the integrated oral human
pharmacokinetic model, it is unlikely that the haematological
effects are inherent to the MT moiety itself, in the dosage range
required for anti-dementia activity. At higher oral doses, shown
for example in the rat study above, it is clear that haematological
adverse would be seen, but it is unlikely that these doses would be
reached in clinical usage of MTC-based forms of a medicinal
product.
[0628] Given the dose-response relationship observed in the rat
study discussed above in the section Relationship Between Cognitive
and Haematological Activity, the expected effect on total red cell
count can be calculated, as shown in FIG. 36. As can be seen, the
expected haematological side effects as indexed by decline in red
cell count is expected to be negligible.
Feasibility of Delayed Release Formulation of the L-MTx Form of the
Methylthioninium Moiety
[0629] Whereas, for the reasons discussed above, it would not be
feasible to generate a delayed-release formulation of an MTC-based
medicinal product, this would not be the case for an L-MTx-based
form of the methylthioninium moiety. This is because the leuco-form
of the methylthioninium cannot dimerise. This is because it is not
a `flat` molecule (unlike Ox-MT), and it has not charge which
permits stabilisation of the dimeric form by charge
neutralisation.
[0630] Therefore, it is likely that a delayed-release formulation
of the L-MTx-based form of the methylthioninium moiety would be
feasible without encountering the adverse consequences of delayed
absorption. This could be created in a once-daily dosage form.
REFERENCES FOR EXAMPLES 1-8
[0631] Birks, J. (2006) Cholinesterase inhibitors for Alzheimer's
disease. Cochrane Database Syst. Rev. (1): CD005593. [0632]
DiSanto, A. R., Wagner, J. G. (1972a) Pharmacokinetics of highly
ionized drugs. I: Methylene blue--whole blood, urine and tissue
assays. Journal of Pharmaceutical Sciences, 61:598-602. [0633]
DiSanto, A. R., Wagner, J. G. (1972b) Pharmacokinetics of highly
ionized drugs. II. Methylene blue--absorption, metabolism, and
excretion in man and dog after oral administration. Journal of
Pharmaceutical Sciences, 61:1086-1090. [0634] DiSanto, A. R.,
Wagner, J. G. (1972c) Pharmacokinetics of highly ionized drugs.
III. Methylene blue--blood levels in the dog and tissue levels in
the rat following intravenous administration. Journal of
Pharmaceutical Sciences, 61:1090-1094. [0635] Gunics, G.,
Motohashi, N., Amaral, L., Farkas, S. & Molnar, J. (2000)
Interaction between antibiotics and non-conventional antibiotics on
bacteria. International Journal of Antimicrobial Agents 14:239-42.
[0636] Kristiansen, J. E., Amaral, L. (1997) The potentional
management of resistant infection with non-antibiotics. Journal of
Antimicrobial Chemotherapy, 40:319-327. [0637] Lewis, G. N.,
Bigeleisen, J. (1943) Methylene blue and other indicators in
general acids. The acidity function: J. Amer. Chem. Soc.,
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Reduction and uptake of methylene blue by human erythrocytes. Am.
J. Physiol. Cell Physiol., 286:C1390-C1398. [0639] Merker, M. P.,
Bongard, R. D., Kettenhofen, N. J., Okamoto, Y., Dawson, C. A.
(2002) Intracellular redox status affects transplasma membrane
electron transport in pulmonary arterial endothelial cells. Am. J.
Physiol. Lung Cell. Mol. Physiol., 282:L36-L43. [0640] Merker, M.
P., Olson, L. E., Bongard, R. D., Patel, M. K., Linehan, J. H.,
Dawson, C. A. (1998) Ascorbate-mediated transplasma membrane
electron transport in pulmonary arterial endothelial cells. Am. J.
Physiol., 274:L685-L693. [0641] Peter, C., Hongwan, D., Kupfer, A.,
Lauterburg, B. H. (2000) Pharmacokinetics and organ distribution of
intravenous and oral methylene blue. Eur. J. Clin. Pharmacol., 56:
247-250. [0642] Rabinowitch, E., Epstein, L. (1941) Polymerization
of dyestuffs in solution. Thionine and methylene blue. J. Am. Chem.
Soc. 63:69-78. [0643] Spencer, W., Sutter, J. R. (1979) Kinetic
study of the monomer-dimer equilibrium of methylene blue in aqueous
suspension. J. Phys. Chem., 83:1573-1576. [0644] Selkoe, D. J.
(2004) Cell biology of protein misfolding: the examples of
Alzheimer's and Parkinson's diseases. Nat. Cell. Biol.,
6:1054-1061. [0645] Moody, J. P., Allan, S. M., Smith, A. H.,
Naylor, G. J. (1989). Methylene blue excretion in depression. Biol.
Psychiatry; 26:850-852. [0646] Rengelshausen, J., Burhenne, J.,
Frohlich, M., Tayrouz, Y., Singh, S. K., Riedel, K.-D., Muller, O.,
Hoppe-Tichy, T., Haefeli, W. E., Mikus, G. & Walter-Sack, I.
(2004) Pharmacokinetic interaction of chloroquine and methylene
blue combination against malaria. European Journal of Clinical
Pharmacology 60:709-715. [0647] Wischik, C. M., Lai, R. Y. K.,
Harrington, C. R. (1997) Modelling prion-like processing of tau
protein in Alzheimer's disease for pharmaceutical development. In
Microtubule-Associated Proteins: Modifications in Disease. (eds. J.
Avila, R. Brandt, & K. S. Kosik) Harwood Academic Publishers,
Amsterdam, 185-241. [0648] Gibaldi, M. and Perrier, D. (1982)
Pharmacokinetics. 2nd edn. Marcel Dekker Inc., New York. [0649]
Braak, H., Braak, E. (1991) Neuropathological staging of
Alzheimer-related changes. Acta Neuropathologica 82:239-259. [0650]
Kuriloff, D. B., Sanborn, K. V. (2004) Rapid intraoperative
localization of parathyroid glands utilizing methylene blue
infusion. Otolaryngology--Head & Neck Surgery 131:616-622.
Example 9
Chemical Synthesis of Stable Crystalline Reduced Form DAPTZ
Compounds
[0651] The following disclosure generally corresponds to that in
prior filed, unpublished, application PCT/GB2007/001103
(specifically incorporated by reference) but is included herein for
completeness only.
[0652] For example, a suitable phenothiazine may be converted to
the corresponding 3,7-dinitro-phenothiazine, for example, using
sodium nitrite with acetic acid and chloroform. The ring amino
group may then be protected, for example, as the acetate, for
example, using acetic anhydride and pyridine. The nitro groups may
then be reduced to amino groups, for example, using tin (II)
chloride with ethanol. The amino groups may then be substituted,
for example, disubstituted, for example, methyl disubstituted, for
example, using methyl iodide, sodium hydroxide, DMSO, and
tetra-n-butyl ammonium bromide. The amino group may then be
deprotected, for example, the N-acetyl group may be removed, for
example, using concentrated aqueous hydrochloride acid. The
corresponding salt is then prepared, for example, using
concentrated aqueous hydrochloric acid, for example, at the same
time as deprotection. An example of such a method is illustrated in
the following scheme.
##STR00012##
[0653] Thus, another aspect of the invention pertains to a method
of preparing a 3,7-diamino-10H-phenothiazine compound of the
following formula, for use in the methods of treatment described
above:
##STR00013##
[0654] wherein R.sup.1, R.sup.9, R.sup.3NA, R.sup.3NB, R.sup.7NA,
R.sup.7NB, HX.sup.1 and HX.sup.2 are as defined herein (for
example, where HX.sup.1 and HX.sup.2 are each HCl), comprising the
step of: [0655] (vi) salt formation (SF).
[0656] In one embodiment, the method comprises the steps of: [0657]
(v) ring amino deprotection (DP); and [0658] (vi) salt formation
(SF).
[0659] In one embodiment, the method comprises the steps of: [0660]
(iv) amine substitution (AS), [0661] optional (v) ring amino
deprotection (DP), and [0662] (vi) salt formation (SF).
[0663] In one embodiment, the method comprises the steps of [0664]
(iii) nitro reduction (NR), [0665] (iv) amine substitution (AS),
[0666] (v) ring amino deprotection (DP), and [0667] (vi) salt
formation (SF).
[0668] In one embodiment, the method comprises the steps of [0669]
optional (ii) ring amino protection (AP), [0670] (iii) nitro
reduction (NR), [0671] (iv) amine substitution (AS), [0672] (v)
ring amino deprotection (DP), and [0673] (vi) salt formation
(SF).
[0674] In one embodiment, the method comprises the steps of [0675]
(i) nitration (NO), [0676] (ii) ring amino protection (AP), [0677]
(iii) nitro reduction (NR), [0678] (iv) amine substitution (AS),
[0679] (v) ring amino deprotection (DP), and [0680] (vi) salt
formation (SF).
[0681] In one embodiment, the steps are performed in the order
listed (i.e., any step in the list is performed at the same time
as, or subsequent to, the preceding step in the list).
[0682] In one embodiment, the step of (v) ring amino deprotection
(DP) and the step of (vi) salt formation (SF) are performed
simultaneously (i.e., as one step).
[0683] In one embodiment, the nitration (NO) step is: [0684] (i)
nitration (NO), wherein a 10H-phenothiazine is converted to a
3,7-dinitro-10H-phenothiazine, for example:
##STR00014##
[0685] In one embodiment, nitration is performed using a nitrite,
for example, sodium nitrite, for example, sodium nitrite with
acetic acid and chloroform. In one embodiment, R.sup.10 is --H.
[0686] In one embodiment, the ring amino protection (AP) step is:
[0687] (ii) ring amino protection (AP), wherein the ring amino
group (--NH--) of a 3,7-dinitro-10H-phenothiazine is converted to a
protected ring amino group (--NR.sup.prot), for example:
##STR00015##
[0688] In one embodiment, ring amino protection is achieved as an
acetate, for example, using acetic anhydride, for example, using
acetic anhydride and pyridine.
[0689] In one embodiment, the nitro reduction (NR) step is: [0690]
(iii) nitro reduction (NR), wherein each of the nitro (--NO.sub.2)
groups of a protected 3,7-dinitro-10H-phenothiazine is converted to
an amino (--NH.sub.2) group, for example:
##STR00016##
[0691] In one embodiment, nitro reduction may be performed using,
for example, tin (II) chloride, for example, tin (II) chloride with
ethanol.
[0692] In one embodiment, the amine substitution (AS) step is:
[0693] (iv) amine substitution (AS), wherein each of the amino
(--NH.sub.2) groups of a protected 3,7-diamino-10H-phenothiazine is
converted to disubstituted amino group, for example:
##STR00017##
[0694] In one embodiment, amine substitution is performed using an
alkyl halide, for example, an alkyl iodide, for example, methyl
iodide, for example, methyl iodide with sodium hydroxide, DMSO, and
tetra-n-butyl ammonium bromide.
[0695] In one embodiment, the ring amino deprotection (DP) step is:
[0696] (v) ring amino deprotection (DP), wherein the protecting
group, R.sup.Prot, is removed, for example:
##STR00018##
[0697] In one embodiment, ring amino deprotection may be performed
using acid, for example, hydrochloric acid, for example,
concentrated aqueous hydrochloric acid.
[0698] In one embodiment, the step is: [0699] (vi) salt formation
(SF), wherein the corresponding salt is formed, for example:
##STR00019##
[0700] In one embodiment, salt formation may be performed using
acid, for example, hydrochloric acid, for example, concentrated
aqueous hydrochloric acid.
[0701] In one embodiment, the steps of ring amine deprotection and
salt formation are performed simultaneously (i.e., as one step),
for example, compound (1) is formed from compound (2) in one
step.
[0702] In another approach, a suitable thioninium chloride (for
example, methylthioninium chloride, MTC) is converted to the
corresponding halide, for example, by reaction with potassium
iodide, for example, aqueous potassium iodide. The resulting
thioninium iodide is then reduced, for example, with ethyl iodide
and ethanol, and the corresponding salt formed. A similar method is
described in Drew, H. D. K, and Head, F. S. H., "Derivatives of
Methylene-blue," Journal of the Chemical Society, 1933, pp.
248-253. An example of such a method is illustrated in the
following scheme.
##STR00020##
[0703] Thus, another aspect of the invention pertains to a method
of preparing a DAPTZ compound of the following formula, for use in
the methods of treatment described above:
##STR00021##
[0704] wherein R.sup.1, R.sup.9, R.sup.3NA, R.sup.3NB, R.sup.7NA,
R.sup.7NB, HX.sup.1 and HX.sup.2 are as defined herein (for
example, where HX.sup.1 and HX.sup.2 are each HI), comprising the
step of: [0705] (ii) reduction and iodide salt formation
(RISF).
[0706] In one embodiment, the method comprises the steps of: [0707]
(i) iodide exchange (1E); and [0708] (ii) reduction and iodide salt
formation (RISE).
[0709] In one embodiment, the steps are performed in the order
listed (i.e., any step in the list is performed at the same time
as, or subsequent to, the preceding step in the list).
[0710] In one embodiment, the iodide exchange (1E) step is: [0711]
(i) iodide exchange (1E), wherein a 3,7-di(disubstituted
amino)-thioninium salt is converted to the corresponding
3,7-di(disubstituted amino)-thioninium iodide, for example (where
Y.sup.- is an anionic counter ion, for example, halide, for
example, chloride or bromide):
##STR00022##
[0712] In one embodiment, iodide exchange (IE) is achieved by
reaction with potassium iodide, for example, aqueous potassium
iodide.
[0713] In one embodiment, the reduction and iodide salt formation
(RISF) step is: [0714] (ii) reduction and iodide salt formation
(RISF), wherein a 3,7-di(disubstituted amino)-thioninium iodide is
reduced and converted to the corresponding
3,7-diamino-10H-phenothiazine iodide compound, for example:
##STR00023##
[0715] In one embodiment, reduction and iodide salt formation
(RISF) is achieved by reaction with ethyl iodide, for example,
ethyl iodide and ethanol.
[0716] In another approach, an appropriate thioninium salt, for
example, ethyl thioninium semi zinc chloride, is simultaneously
reduced and the ring amino group protected, for example, by
reaction with phenylhydrazine, ethanol, acetic anhydride, and
pyridine. The corresponding salt may then be prepared, for example,
using concentrated aqueous hydrochloric acid, for example, at the
same time as deprotection. An example of such a method is
illustrated in the following scheme.
##STR00024##
[0717] Thus, another aspect of the invention pertains to a method
of preparing a 3,7-diamino-10H-phenothiazine (DAPTZ) compound of
the following formula:
##STR00025##
[0718] wherein R.sup.1, R.sup.9, R.sup.3NA, R.sup.3NB, R.sup.7NA,
R.sup.7NB, HX.sup.1 and HX.sup.2 are as defined herein (for
example, where HX.sup.1 and HX.sup.2 are each HI), comprising the
step of:
[0719] comprising the step of: [0720] (iv) salt formation (SF).
[0721] In one embodiment, the method comprises the steps of [0722]
(iii) ring amino deprotection (DP), and [0723] (iv) salt formation
(SF).
[0724] In one embodiment, the method comprises the steps of [0725]
(ii) ring amino protection (AP), [0726] (iii) ring amino
deprotection (DP), and [0727] (iv) salt formation (SF).
[0728] In one embodiment, the method comprises the steps of [0729]
(i) reduction (RED) [0730] (ii) ring amino protection (AP), [0731]
(iii) ring amino deprotection (DP), and [0732] (iv) salt formation
(SF).
[0733] In one embodiment, the steps are performed in the order
listed (i.e., any step in the list is performed at the same time
as, or subsequent to, the preceding step in the list).
[0734] In one embodiment, the step of (i) reduction (RED) and the
step of (ii) ring amino protection (AP) are performed
simultaneously (i.e., as one step).
[0735] For example, in one embodiment, the combined reduction (RED)
step and ring amino protection (AP) step is: [0736] (i) reduction
(RED) and ring amino protection (AP), wherein a
3,7-di(disubstituted amino)-thioninium salt is reduced to give the
corresponding 3,7-di(disubstituted amino)-10H-phenothiazine, and
the ring amino group (--NH--) of the 3,7-di(disubstituted
amino)-10H-phenothiazine is converted to a protected ring amino
group (--R.sup.prot) to give the corresponding protected
3,7-di(disubstituted amino)-10H-phenothiazine, for example:
##STR00026##
[0737] In one embodiment, Y represents Cl.sup.-.
[0738] In one embodiment, the combined reduction (RED) step and
ring amino protection (AP) step is achieved using phenylhydrazine
and acetic anhydride, for example, phenylhydrazine, ethanol, acetic
anhydride, and pyridine.
[0739] In one embodiment, the step of (iii) ring amino deprotection
(DP) and the step of (iv) salt formation (SF) are performed
simultaneously (i.e., as one step).
[0740] For example, in one embodiment, the combined ring amino
deprotection (DP) step and salt formation (SF) step is: [0741] (ii)
ring amino deprotection (DP) and salt formation (SF), wherein the
protecting group of a protected 3,7-di(disubstituted
amino)-10H-phenothiazine is removed to give a 3,7-di(disubstituted
amino)-10H-phenothiazine, and the corresponding salt is formed, for
example:
##STR00027##
[0742] In one embodiment, the combined ring amino deprotection (DP)
step and salt formation (SF) step may be performed using acid, for
example, hydrochloric acid, for example, concentrated aqueous
hydrochloric acid.
[0743] In a similar approach, an appropriate thioninium chloride
(e.g., methyl thioninium chloride, ethyl thioninium chloride) is
first reduced and acetylated to give the corresponding
1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone, for example,
by reaction with hydrazine (NH.sub.2NH.sub.2), methyl hydrazine
(MeNHNH.sub.2), or sodium borohydride (NaBH.sub.4); and acetic
anhydride ((H.sub.3CCO).sub.2O); for example, in the presence of a
suitable base, for example, pyridine (C5H5N) or Hunig's base
(diisopropylethylamine, C.sub.8H.sub.19N), for example, in a
suitable solvent, for example, ethanol or acetonitrile. The reduced
and acetylated compound is then deprotected (by removing the acetyl
group), for example, by reaction with a suitable halic acid, for
example, hydrochloric acid or hydrobromic acid, in a suitable
solvent, for example, ethanol, and optionally with the addition of
a suitable ether, for example, diethyl ether.
[0744] Specific examples are as follows:
CHEMICAL SYNTHESIS
Synthesis 1
3-Nitro-10H-phenothiazine
##STR00028##
[0746] Sodium nitrite (20.00 g, 210 mmol) was added to a mixture of
10H-phenothiazine (20.00 g, 50 mmol), chloroform (100 cm.sup.3),
and acetic acid (20 cm.sup.3), and the mixture was stirred for 1
hour at room temperature. Acetic acid (20 cm.sup.3) was then added
and the mixture was stirred for a further 18 hours. The suspension
was filtered and washed with acetic acid, ethanol, water, and
finally ethanol to give a purple/brown solid. The residue was
dissolved in hot DMF and allowed to cool before filtering the
di-nitro compound as a purple solid. Concentration of the DMF
solution and washing the precipitate with water and methanol gave
the title mono-nitro compound (15 g, .about.50%) as a brown solid;
.nu..sub.max (KBr)/cm.sup.-1: 3328 (NH), 3278 (NH), 3229 (NH), 3119
(CH), 3049 (CH), 1557 (NO.sub.2), 1531 (NO.sub.2); .delta..sub.H
(250 MHz; DMSO): 6.64 (5H, m, ArH), 7.68 (1H, d, J 2.5, ArH),
7.79-7.84 (1H, dd, J2.75, 6.5, ArH); .delta..sub.C (62.9 MHz;
DMSO): 113.3 (ArC), 115.3 (ArC), 116.9 (ArC), 121.8 (ArC), 123.6
(ArC), 123.7 (ArC), 124.6 (ArC), 126.4 (ArC), 128.1 (ArC), 138.8
(ArC), 141.0 (ArC), 147.8 (ArC).
Synthesis 2
3,11-Dinitro-10H-phenothiazine
##STR00029##
[0748] The procedure for the synthesis of 3-nitro-10H-phenothiazine
was followed using 3-nitro-10H-phenothiazine (10.00 g, 41 mmol),
chloroform (40 cm.sup.3), acetic acid (2.times.10 cm.sup.3), and
sodium nitrite (11.86 g, 173 mmol). The residue obtained was
recrystallised from DMF to yield the title di-nitro compound (6.60
g 56%) as purple needles; .nu..sub.max (KBr)/cm.sup.-1: 3331 (NH),
3294 (NH), 3229 (NH), 3101 (CH), 3067 (CH), 1602 (NO.sub.2), 1558
(NO.sub.2); .delta..sub.H (250 MHz; DMSO): 6.73-6.76 (2H, d, J 9,
ArH), 7.78 (2H, s, ArH), 7.89-7.85 (2H, d, J 9, ArH).
Synthesis 3
1-(3,7-Dinitro-phenothiazin-10-yl)-ethanone
##STR00030##
[0750] A solution of 3,11-dinitro-10H-phenothiazine (3.00 g, 10.37
mmol), acetic anhydride (15.88 g, 155.50 mmol), and pyridine (30
cm.sup.3) was stirred at reflux for 18 hours. The warm solution was
then carefully poured over ice water. A precipitate formed and was
filtered, dissolved in dichloromethane, dried over magnesium
sulphate, filtered, and concentrated to give a brown/orange solid,
which was purified by column chromatography (SiO.sub.2, ethyl
acetate: petroleum ether, 2:3, loaded as a dichloromethane
solution) to give the title compound (2.46 g, 71%) as a light
yellow solid which can be recrystallised from acetone to give light
yellow needles; .nu..sub.max (KBr)/cm.sup.-1: 3091 (CH), 3063 (CH),
1680 (C.dbd.O), 1575 (NO.sub.2), 1510 (NO.sub.2); .delta..sub.H
(250 MHz; CDCl.sub.3): 2.28 (3H, s, CH.sub.3), 7.65-7.69 (2H, d, J
9, ArH), 8.22-8.26 (2H, dd, J 2.75, 8.75, ArH), 8.33-8.32 (2H, d, J
2.5, ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3): 168.2 (C.dbd.O),
146.3 (ArC), 143.3 (ArC), 133.6 (ArC), 127.8 (ArC), 123.4 (ArC),
122.9 (ArC), 23.1 (CH.sub.3); m/z (ES) 331.0 (80%, [M].sup.+).
Synthesis 4
1-(3,7-Diamino-phenothiazin-10-yl)-ethanone
##STR00031##
[0752] A mixture of 1-(3,7-dinitro-phenothiazin-10-yl)-ethanone (2
g, 6.04 mmol), tin (II) chloride dihydrate (14.17 g, 62.8 mmol),
and ethanol (50 cm.sup.3) was heated to reflux and stirred at this
temperature for 5 hours. The mixture was then cooled to room
temperature and poured over ice water. The pH was adjusted to 7
with 5% sodium hydrogen carbonate before the product was extracted
with ethyl acetate (3.times.50 cm.sup.3). The extracts were washed
with brine and dried over magnesium sulphate, filtered, and
concentrated to give the title compound (1.64 g, 100%) as a purple
blue solid; .nu..sub.max (KBr)/cm.sup.-1: 3445 (NH), 3424 (NH),
3368 (NH), 3322 (NH), 3203 (NH), 3054 (CH), 2995 (CH), 1706
(C.dbd.O), 1650 (NO.sub.2). 1590 (NO.sub.2); .delta..sub.H (250
MHz; CDCl.sub.3): 2.01 (3H, s, CH.sub.3), 5.09-5.43 (4H, brd s,
NH), 6.47-6.51 (2H, dd, J 1.5, 8.25, ArH), 6.61 (2H, s, ArH),
7.11-7.15 (2H, d, J 8, ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3):
169.1 (C.dbd.O), 147.2 (ArC), 128.1 (ArC), 127.6 (ArC), 127.3
(ArC), 112.3 (ArC), 111.5 (ArC), 22.6 (CH.sub.3); m/z (ES) 293.9
(95%, [M+H, Na].sup.+), 272.0 (20%, [M+H].sup.+) 227.9 (100%, [M+H,
-Ac].sup.+).
Synthesis 5
3,7-Diamino-phenothiazine bis(hydrogen chloride) (B4)
##STR00032##
[0754] 1-(3,7-Diamino-phenothiazin-10-yl)-ethanone (0.25 g, 0.921
mmol) was dissolved in aqueous hydrochloric acid (5 N, 10 cm.sup.3)
and the solution was heated to reflux and stirred for 30 minutes.
Concentration of the reaction mixture gave the title compound as a
light blue solid. .delta..sub.H (250 MHz; D.sub.2O): 6.60 (2H, brd
d, ArH), 7.07 (4H, brd s, ArH).
Synthesis 6
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00033##
[0756] 1-(3,7-Diamino-phenothiazin-10-yl)-ethanone (0.25 g 0.92
mmol) was dissolved in DMSO (3 cm.sup.3). Toluene (10 cm.sup.3),
iodomethane (1.96 g, 13.8 mmol), tetrabutylammoniun bromide (50
mg), and finally aqueous sodium hydroxide solution (50%, 1.25
cm.sup.3) were added. The mixture was stirred at room temperature
for 2 hours. Additional aqueous sodium hydroxide (50%, 1.25
cm.sup.3) and iodomethane (1.96 g, 13.8 mmol) were then added. The
mixture was allowed to stir for a further 3 hours at room
temperature before a third aliquot of aqueous sodium hydroxide
(50%, 1.25 cm.sup.3) and iodomethane (1.96 g, 13.8 mmol) were added
and the mixture stirred for a further 18 hours. The thick
suspension was washed with water (3.times.75 cm.sup.3) and the
toluene extract collected. The water was extracted with
dichloromethane (3.times.50 cm.sup.3) and the extracts combined
with the toluene, and dried over magnesium sulphate, filtered, and
concentrated to give a deep purple solid. The residue was purified
by column chromatography (SiO.sub.2, ethyl acetate: petroleum
ether, 2:3, loaded as a dichloromethane solution) to give the title
compound product (0.12 g, 40%) as a light purple solid;
.nu..sub.max (KBr)/cm.sup.-1: 2910 (CH), 2876 (CH), 2856 (CH), 2799
(CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3): 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3): 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3).
Synthesis 7
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride) (B3)
##STR00034##
[0758] 1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone (0.5
g, 1.84 mmol) was dissolved in aqueous hydrochloric acid (5 N, 15
cm.sup.3), and the solution was heated to reflux temperature and
stirred for 30 minutes. Concentration of the reaction mixture gave
the title compound as a green/blue solid; .delta..sub.H (250 MHz;
D.sub.2O): 3.18 (12H, s, NCH.sub.3), 6.67 (2H, d, J 8.5, ArH), 7.16
(4H, brd s, ArH); .delta..sub.C(62.9 MHz; D.sub.2O): 144.3 (ArC),
138.9 (ArC), 122.4 (ArC), 120.8 (ArC), 120.7 (ArC), 117.6 (ArC),
48.9 (NCH.sub.3).
Synthesis 8
Methylthioninium Iodide
##STR00035##
[0760] To a round bottom flask was added methylthioninium chloride
(MTC, Methylene Blue) (2 g, 6.25 mmol) and water (50 cm.sup.3) and
the mixture stirred for 10 minutes or until the solid dissolved.
Potassium iodide (1.56 g, 9.4 mmol) was then added to the mixture
and a green black suspension formed. The reaction was heated to
boiling and allowed to cool naturally giving the title compound
(2.03 g, 79%) as bright green needles. Anal. Calcd for
C.sub.16H.sub.18N.sub.3SI: C, 46.72; H, 4.41; N, 10.22; S, 7.80;
1,30,85. Found: C, 46.30; H, 4.21; N, 10.14; S, 7.86; I, 29.34.
Synthesis 9
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
iodide) (B6)
##STR00036##
[0762] To a round bottom flask was added methylthioninium iodide (2
g, 4.86 mmol), ethanol (100 cm.sup.3) and ethyl iodide (75.8 g, 486
mmol) and the mixture was heated at reflux for 18 hours where the
colour changed from green/blue to brown with a yellow precipitate.
Once cooled to room temperature, the mixture was filtered and
washed with diethylether (20 cm.sup.3) to give the title compound
(1.99 g, 76%) as a light green solid. .delta..sub.H (250 MHz;
D.sub.2O): 3.20 (12H, s, NCH.sub.3), 6.76 (2H, d, J 8.5, ArH), 7.22
(2H, brd s, ArH); .delta..sub.C (62.9 MHz; D.sub.2O): 145.0 (ArC),
139.3 (ArC), 122.6 (ArC), 121.1 (ArC), 120.9 (ArC), 117.9 (ArC),
48.9 (NCH.sub.3).
Synthesis 10
1-(3,7-Bis-diethylamino-phenothiazin-10-yl)-ethanone
##STR00037##
[0764] To a dry 25 cm.sup.3 round bottom flask was added
ethylthioninium zinc chloride (0.5 g, 1.13 mmol) and ethanol (10
cm.sup.3). Phenylhydrazine (0.134 g, 1.24 mmol) was then added
dropwise under an atmosphere of nitrogen. The mixture was stirred
25.degree. C. for 1 hour and concentrated under high vacuum.
Pyridine (50 cm.sup.3) and acetic anhydride was added and the
mixture stirred for 18 hours at 60.degree. C. The solution was
opened to ice/water (250 cm.sup.3) and the organics were extracted
into ethyl acetate (3.times.50 cm.sup.3). The extracts were washed
with saturated copper sulphate solution and dried over magnesium
sulphate, filtered, and concentrated to give the crude product as a
brown oil, which was purified using flash column chromatography
with an eluent of 40% ethylacetate: 60% petroleum spirit
40-60.degree. C. and silica 40-63.mu. 60{acute over (.ANG.)} to
give the title compound (0.18 g, 41%) as a green glassy solid.
.delta..sub.H (250 MHz; CDCl.sub.3): 7.0-7.5 (2H, brds, ArH), 6.64
(2H, s, ArH), 6.52 (2H, d, ArH), 3.35 (8H, q, 7, NCH.sub.2), 2.18
(3H, s, CH.sub.3), 1.16 (12H, t, 7, CH.sub.3); .delta..sub.C (62.9
MHz; CDCl.sub.3): 12.5 (CH.sub.3), 22.9 (CH.sub.3), 44.6
(NCH.sub.2), 110.1 (ArC), 127.4 (ArC), 146.5 (ArC), 170.2
(C.dbd.O).
Synthesis 11
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)
##STR00038##
[0766] To a 25 cm.sup.3 round bottom flask was added
3,7-diethylamino-10-acetyl-phenothiazine (0.125 g, 0.33 mmol) and
aqueous hydrochloric acid (5 M, 5 cm.sup.3). The mixture was heated
at 100.degree. C. for 2 hours before cooling to room temperature
and was concentrated to give the title compound (0.11 g, 81%) as a
yellow green glassy solid. .delta..sub.H (250 MHz; CD.sub.3OD):
7.07 (4H, brd, ArH), 6.65 (2H, brd, ArH), 3.35 (8H, brd,
NCH.sub.2), 0.97 (12H, brd, CH.sub.3); .delta..sub.C (62.9 MHz;
CD.sub.3OD): 10.8 (CH.sub.3), 55.1 (NCH.sub.2), 116.6 (ArC), 120.4
(ArC), 121.5 (ArC), 123.6 (ArC), 132.6 (ArC), 144.5 (ArC).
Synthesis 12
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00039##
[0768] Synthesis using methylhydrazine/pyridine in two pots. To a
250 cm.sup.3 round bottom flask placed under an atmosphere of argon
was added methylthioninium chloride trihydrate (26.74 mmol, 10 g),
ethanol (100 cm.sup.3) and methylhydrazine (58.83 mmol, 2.71 g).
The mixture was heated to 40.degree. C. and stirred for 2 hours.
The yellow/green suspension was cooled to 5.degree. C. and filtered
under argon, washed with ethanol (20 cm.sup.3) and dried to give
leuco-methylene blue as a light green solid. To the leuco product
was added acetic anhydride (40 cm.sup.3) and pyridine (10 cm.sup.3)
and the solution was heated at 100.degree. C. for 18 hours. The
cooled mixture was then poured carefully over ice water while
stirring to give a precipitate, which was filtered, washed with
water, and dried at 60.degree. C. for 2 hours to yield the title
compound (5.82 g, 66%) as a light brown solid. Mp 137.degree. C.;
v.sub.max(KBr)/cm.sup.-1 2910 (CH), 2876 (CH), 2856 (CH), 2799
(CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3); m/z (ES) 284.2 (100%, [M-OAc].sup.+), 328.1 (15%,
[M+H].sup.+), 350.1 (41%, [M+Na].sup.+).
Synthesis 13
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00040##
[0770] Synthesis using methylhydrazine/Hunig's base in one pot. To
a 5000 cm.sup.3 reactor vessel under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (0.54 mol, 200 g) and
acetonitrile (1000 cm.sup.3). Methylhydrazine (1.07 mol, 49.36 g)
was added dropwise at 1.5 mL per minute. The temperature of the
mixture increased to 32.degree. C. and was stirred for 20 minutes.
The yellow/green suspension had acetic anhydride (5.35 mol, 541 g)
added and then Hunig's base (diisopropylethylamine) (1.55 mol, 200
g) was added. The mixture was heated at 90.degree. C. for 2 hours.
The cooled mixture was then poured carefully into ice water (2000
cm.sup.3) in ten 200 cm.sup.3 portions while stirring to give a
precipitate. The precipitate was stirred for 45 minutes before it
was filtered, washed with water (3.times.250 cm.sup.3). and air
dried for 30 minutes. The crude material was crystallised from hot
ethanol (2750 cm.sup.3) to yield the title compound (112.1 g, 64%)
as a light grey solid. Mp 137.degree. C.; v.sub.max(KBr)/cm.sup.-1
2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659 (C.dbd.O), 1596
(NO.sub.2), 1502 (NO.sub.2); .delta..sub.H (250 MHz; CDCl.sub.3)
2.16 (3H, s, CH.sub.3), 2.93 (12H, s, NCH.sub.3), 6.59-6.62 (2H, d,
J 8.5, ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s,
ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9
(ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7
(ArC), 40.7 (NCH.sub.3), 22.9 (CH.sub.3); m/z (ES) 284.2 (100%,
[M-OAc].sup.+), 328.1 (15%, [M+H].sup.+), 350.1 (41%,
[M+Na].sup.+).
Synthesis 14
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00041##
[0772] Synthesis using methylhydrazine/pyridine in one pot. To a
250 cm.sup.3 round bottom flask under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (26.74 mmol, 10 g) and
acetonitrile (50 cm.sup.3). Methylhydrazine (53.5 mmol, 2.46 g) was
added in four equal portions over a 30 minutes time period. The
temperature of the mixture was maintained at 35.degree. C. with a
cold water bath and was stirred for 30 minutes. The yellow/green
suspension had acetic anhydride (267 mmol, 27.3 g) and pyridine
(80.2 mmol, 6.35 g) was added. The mixture was heated at 90.degree.
C. for 2 hours. The cooled mixture was then poured carefully into
ice water (200 cm.sup.3) in ten equal portions while stirring to
give a precipitate. The precipitate was stirred for 30 minutes
before it was filtered, washed with water (3.times.50 cm.sup.3) and
air dried for 30 minutes. The crude material was crystallised from
hot ethanol (120 cm.sup.3) to yield the title compound (5.97 g,
68%) as a light grey solid. Mp 137.degree. C.;
v.sub.max(KBr)/cm.sup.-1 2910 (CH), 2876 (CH), 2856 (CH), 2799
(CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3); m/z (ES) 284.2 (100%, [M-OAc].sup.+), 328.1 (15%,
[M+H].sup.+), 350.1 (41%, [M+Na].sup.+).
Synthesis 15
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00042##
[0774] Synthesis using sodium borohydride/pyridine in one pot. To a
500 cm.sup.3 round bottom flask under an atmosphere of nitrogen was
added methylthioninium chloride trihydrate (0.134 mol, 50 g) and
acetonitrile (250 cm.sup.3). Sodium borohydride (0.174 mol, 6.6 g)
was added in four equal portions over a 30 minute time period. The
temperature of the mixture was maintained at 35.degree. C. with a
cold water bath and was stirred for 30 minutes. The yellow/green
suspension had acetic anhydride (0.535 mol, 55 g) and pyridine
(0.174 mol, 13.76 g) added. The mixture was heated at 90.degree. C.
for 2 hours. The cooled mixture was then poured carefully into ice
water (250 cm.sup.3) in ten equal portions while stirring to give a
precipitate. The precipitate was stirred for 30 minutes before it
was filtered, washed with water (3.times.50 cm.sup.3), and air
dried for 30 minutes. The crude material was crystallised from hot
ethanol (500 cm.sup.3) to yield the title compound (26.7 g, 61%) as
a light grey solid. Mp 137.degree. C.; v.sub.max(KBr)/cm.sup.-1
2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659 (C.dbd.O), 1596
(NO.sub.2), 1502 (NO.sub.2); .delta..sub.H (250 MHz; CDCl.sub.3)
2.16 (3H, s, CH.sub.3), 2.93 (12H, s, NCH.sub.3), 6.59-6.62 (2H, d,
J 8.5, ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s,
ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9
(ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7
(ArC), 40.7 (NCH.sub.3), 22.9 (CH.sub.3); m/z (ES) 284.2 (100%,
[M-OAc].sup.+), 328.1 (15%, [M+H].sup.+), 350.1 (41%,
[M+Na].sup.+).
Synthesis 16
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00043##
[0776] Synthesis using sodium borohydride/Hunig's base in one pot.
To a 500 cm.sup.3 round bottom flask under an atmosphere of
nitrogen was added methylthioninium chloride trihydrate (80.2 mmol,
30 g) and acetonitrile (150 cm.sup.3). Sodium borohydride (104
mmol, 3.94 g) was added in four equal portions over a 30 minute
time period. The temperature of the mixture was maintained at
35.degree. C. with a cold water bath and was stirred for 30
minutes. The yellow/green suspension had acetic anhydride (321
mmol, 32.75 g) and Hunig's base (diisopropylethylamine) (120 mmol,
15.55 g) added. The mixture was heated at 90.degree. C. for 2
hours. The cooled mixture was then poured carefully into ice water
(200 cm.sup.3) in ten equal portions while stirring to give a
precipitate. The precipitate was stirred for 30 minutes before it
was filtered, washed with water (3.times.50 cm.sup.3), and air
dried for 30 minutes. The crude material was crystallised from hot
ethanol (300 cm.sup.3) to yield the title compound (13.55 g, 52%)
as a light grey solid. Mp 137.degree. C.; v.sub.max(KBr)/cm.sup.-1
2910 (CH), 2876 (CH), 2856 (CH), 2799 (CH), 1659 (C.dbd.O), 1596
(NO.sub.2), 1502 (NO.sub.2); .delta..sub.H (250 MHz; CDCl.sub.3)
2.16 (3H, s, CH.sub.3), 2.93 (12H, s, NCH.sub.3), 6.59-6.62 (2H, d,
J 8.5, ArH), 6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s,
ArH); .delta..sub.C (62.9 MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9
(ArC), 127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7
(ArC), 40.7 (NCH.sub.3), 22.9 (CH.sub.3); m/z (ES) 284.2 (100%,
[M-OAc].sup.+), 328.1 (15%, [M+H].sup.+), 350.1 (41%,
[M+Na].sup.+).
Synthesis 17
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00044##
[0778] Synthesis using hydrazine monohydrate/pyridine in one pot.
To a 250 cm.sup.3 round bottom flask under an atmosphere of
nitrogen was added methylthioninium chloride trihydrate (26.74
mmol, 10 g) and acetonitrile (50 cm.sup.3). Hydrazine monohydrate
(58.8 mmol, 2.95 g) was added and the mixture was heated to reflux
and stirred for 10 minutes before cooling to 25.degree. C. The
yellow/green suspension had acetic anhydride (424 mmol, 43.3 g) and
pyridine (124 mmol, 9.78 g) added. The mixture was heated at
90.degree. C. for 2 hours. The cooled mixture was then poured
carefully into ice water (100 cm.sup.3) in ten equal portions while
stirring to give a precipitate. The precipitate was stirred for 30
minutes before it was filtered, washed with water (3.times.50
cm.sup.3), and air dried for 30 minutes. The crude material was
crystallised from hot ethanol (100 cm.sup.3) to yield the title
compound (4.87 g, 56%) as a light grey solid. Mp 137.degree. C.;
v.sub.max(KBr)/cm.sup.-1 2910 (CH), 2876 (CH), 2856 (CH), 2799
(CH), 1659 (C.dbd.O), 1596 (NO.sub.2), 1502 (NO.sub.2);
.delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s, CH.sub.3), 2.93
(12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH), 6.69-6.71 (2H,
d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH); .delta..sub.C (62.9
MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC), 127.2 (ArC), 127.1
(ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC), 40.7 (NCH.sub.3),
22.9 (CH.sub.3); m/z (ES) 284.2 (100%, [M-OAc].sup.+), 328.1 (15%,
[M+H].sup.+), 350.1 (41%, [M+Na].sup.+).
Synthesis 18
1-(3,7-Bis-dimethylamino-phenothiazin-10-yl)-ethanone
##STR00045##
[0780] Synthesis using hydrazine monohydrate/Hunig's base in one
pot. To a 250 cm.sup.3 round bottom flask under an atmosphere of
nitrogen was added methylthioninium chloride trihydrate (80.2 mmol,
30 g) and acetonitrile (150 cm.sup.3). Hydrazine monohydrate (176.5
mmol, 8.84 g) was added and the mixture was heated to reflux and
stirred for 10 minutes before cooling to 25.degree. C. The
yellow/green suspension had acetic anhydride (794 mmol, 81.2 g) and
Hunig's base (diisopropylethylamine) (232 mmol, 29.97 g) added. The
mixture was heated at 90.degree. C. for 2 hours. The cooled mixture
was then poured carefully into ice water (400 cm.sup.3) in ten
equal portions while stirring to give a precipitate. The
precipitate was stirred for 30 minutes before it was filtered,
washed with water (3.times.100 cm.sup.3), and air dried for 30
minutes. The crude material was crystallised from hot ethanol (400
cm.sup.3) to yield the title compound (17.15 g, 65%) as a light
grey solid. Mp 137.degree. C.; v.sub.max(KBr)/cm.sup.-1 2910 (CH),
2876 (CH), 2856 (CH), 2799 (CH), 1659 (C.dbd.O), 1596 (NO.sub.2),
1502 (NO.sub.2); .delta..sub.H (250 MHz; CDCl.sub.3) 2.16 (3H, s,
CH.sub.3), 2.93 (12H, s, NCH.sub.3), 6.59-6.62 (2H, d, J 8.5, ArH),
6.69-6.71 (2H, d, J 2.75, ArH), 7.08-7.47 (2H, brd s, ArH);
.delta..sub.C (62.9 MHz; CDCl.sub.3) 170.3 (C.dbd.O), 148.9 (ArC),
127.2 (ArC), 127.1 (ArC), 127.0 (ArC), 110.9 (ArC), 110.7 (ArC),
40.7 (NCH.sub.3), 22.9 (CH.sub.3); m/z (ES) 284.2 (100%,
[M-OAc].sup.+), 328.1 (15%, [M+H].sup.+), 350.1 (41%,
[M+Na].sup.+).
Synthesis 19
3,11-Dinitro-10H-phenothiazine
##STR00046##
[0782] 10H-Phenothiazine (20.00 g, 100 mmol), dichloromethane (100
cm.sup.3) and acetic acid (40 cm.sup.3) had sodium nitrite (20.07
g, 300 mmol) added and the mixture was stirred for 10 minutes at
room temperature. Additional acetic acid (40 cm.sup.3),
dichloromethane (100 cm.sup.3) and sodium nitrite (20.07 g, 300
mmol) were then added. A further 120 cm.sup.3 of acetic acid was
added to try and break up the thick reaction mixture. The mixture
was stirred for 3 hours. The suspension was filtered and washed
with 100 cm.sup.3 each of ethanol, water, and finally ethanol to
give a purple/brown solid. The residue was stirred in hot DMF and
allowed to cool before filtering the dinitro product, which was
washed with ethanol (150 cm.sup.3) and dried to give the title
compound (24.88 g, 86%) as a brown solid; v.sub.max(KBr)/cm.sup.-1
3331 (NH), 3294 (NH), 3229 (NH), 3101 (CH), 3067 (CH), 1602
(NO.sub.2), 1558 (NO.sub.2); .delta..sub.H (250 MHz; DMSO)
6.73-6.76 (2H, d, J 9, ArH), 7.78 (2H, s, ArH), 7.89-7.85 (2H, d, J
9, ArH).
Synthesis 20
1-(3,7-Bis-diethylamino-phenothiazin-10-yl)-ethanone
##STR00047##
[0784] To a 250 cm.sup.3 round bottom flask under an atmosphere of
nitrogen was added ethylthioninium nitrate monohydrate (7.13 mmol,
3 g) and acetonitrile (20 cm.sup.3). Hydrazine monohydrate (16.4
mmol, 0.82 g) was added and the mixture was heated to reflux and
stirred for 10 minutes before cooling to 25.degree. C. The brown
solution had acetic anhydride (114 mmol, 11.65 g) and Hunig's base
(diisopropylethylamine) (21.4 mmol, 2.77 g) was added. The mixture
was heated at 90.degree. C. for 2 hours. The cooled mixture was
then poured carefully into ice water (40 cm.sup.3) in ten equal
portions while stirring to give a precipitate. The precipitate was
stirred for 30 minutes before it was filtered, washed with water
(3.times.25 cm.sup.3) and air dried for 30 minutes. The crude
material was crystallised from hot ethanol (50 cm.sup.3) to yield
the title compound (1.73 g, 63%) as a light grey solid.
.delta..sub.H (250 MHz; CDCl.sub.3) 7.0-7.5 (2H, brds, ArH), 6.64
(2H, s, ArH), 6.52 (2H, d, ArH), 3.35 (8H, q, 7, NCH.sub.2), 2.18
(3H, s, CH.sub.3), 1.16 (12H, t, 7, CH.sub.3); .delta..sub.C (62.9
MHz; CDCl.sub.3) 12.5 (CH.sub.3), 22.9 (CH.sub.3), 44.6
(NCH.sub.2), 110.1 (ArC), 127.4 (ArC), 146.5 (ArC), 170.2
(C.dbd.O).
Synthesis 21
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)
##STR00048##
[0786] To a round bottom flask was added
1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.30
mmol), ethanol (5 cm.sup.3), and hydrochloric acid (37%, 1.3
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, the mixture was concentrated
giving the title compound (0.54 g, 100%) as a light green glass.
.delta..sub.H (250 MHz; CD.sub.3OD) 7.07 (4H, brd, ArH), 6.65 (2H,
brd, ArH), 3.35 (8H, brd, NCH.sub.2), 0.97 (12H, brd, CH.sub.3);
.delta..sub.C (62.9 MHz; CD.sub.3OD) 10.8 (CH.sub.3), 55.1
(NCH.sub.2), 116.6 (ArC), 120.4 (ArC), 121.5 (ArC), 123.6 (ArC),
132.6 (ArC), 144.5 (ArC).
Synthesis 22
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide)
##STR00049##
[0788] To a round bottom flask was added
1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (0.5 g, 1.30
mmol), ethanol (5 cm.sup.3), and hydrobromic acid (48%, 0.75
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, the mixture was concentrated
giving the title compound (0.65 g, 100%) as a light yellow glass.
.delta..sub.H (250 MHz; D.sub.2O) 7.05 (4H, brd, ArH), 6.79 (2H,
brd d, ArH), 3.43 (8H, brd, NCH.sub.2), 1.05 (12H, brd t,
CH.sub.3); .delta..sub.C (62.9 MHz; D.sub.2O) 12.3 (CH.sub.3), 56.2
(NCH.sub.2), 117.9 (ArC), 121.4 (ArC), 122.4 (ArC), 124.5 (ArC),
133.5 (ArC), 145.1 (ArC).
Synthesis 23
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
chloride)
##STR00050##
[0790] To a round bottom flask was added
1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone (1 g, 3.05
mmol), ethanol (10 cm.sup.3), and hydrochloric acid (37%, 3
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, diethyl ether was added while
stirring until a constant turbid solution was obtained. After some
time, a precipitate formed, which was filtered and washed with
diethyl ether (10 cm.sup.3) giving the title compound (0.98 g, 90%)
as a light green solid. Mp (dec) 230.degree. C.; v.sub.max
(KBr)/cm.sup.-1 3500-3229 (NH), 3061 (CH), 3021 (CH), 2948 (CH),
2879 (CH), 2679 (CH), 2601 (CH), 1604 (CH), 1483 (CH), 1318 (CH);
.delta..sub.H (250 MHz; D.sub.2O) 3.18 (12H, s, NCH.sub.3), 6.67
(2H, d, J 8.5, ArH), 7.16 (4H, brd s, ArH); .delta..sub.C (62.9
MHz; D.sub.2O) 144.3 (ArC), 138.9 (ArC), 122.4 (ArC), 120.8 (ArC),
120.7 (ArC), 117.6 (ArC), 48.9 (NCH.sub.3); m/z (ES) 286.1 (100%,
[M-H, 2Cl].sup.+), 285.1 (40%), 284.1 (41%, [M-3H, 2Cl].sup.+).
Synthesis 24
N,N,N',N'-Tetramethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide)
##STR00051##
[0792] To a round bottom flask was added
1-(3,7-bis-dimethylamino-phenothiazin-10-yl)-ethanone (1 g, 3.05
mmol), ethanol (10 cm.sup.3), and hydrobromic acid (48%, 4
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to room temperature, a precipitate formed, which was
filtered and washed with diethyl ether (10 cm.sup.3) giving the
product (1.22 g, 89%) as a light mustard solid. Mp (dec)
230.degree. C.; v.sub.max(KBr)/cm.sup.-1 3500-3229 (NH), 3061 (CH),
3021 (CH), 2948 (CH), 2879 (CH), 2679 (CH), 2601 (CH), 1604 (CH),
1483 (CH), 1318 (CH); .delta..sub.H (250 MHz; D.sub.2O) 3.18 (12H,
s, NCH.sub.3), 6.66 (2H, d, J 8.75, ArH), 7.15 (4H, s, ArH);
.delta..sub.C (62.9 MHz; D.sub.2O) 144.3 (ArC), 138.9 (ArC), 122.4
(ArC), 120.8 (ArC), 120.7 (ArC), 117.6 (ArC), 48.9 (NCH.sub.3).
Synthesis 25
N,N,N',N'-Tetraethyl-10H-phenothiazine-3,7-diamine bis(hydrogen
bromide)
##STR00052##
[0794] To a round bottom flask was added
1-(3,7-bis-diethylamino-phenothiazin-10-yl)-ethanone (1.0 g, 2.60
mmol), methanol (10 cm.sup.3), and hydrobromic acid (48%, 2.94
cm.sup.3) and the solution was heated at 80.degree. C. for 1 hour.
Once cooled to 5.degree. C., the mixture had diethyl ether added,
giving a cloudy solution. The solution was stirred for 30 minutes
and gave the title compound (0.83 g, 63%) as a light yellow solid.
.delta..sub.H (250 MHz; D.sub.2O) 7.05 (4H, brd, ArH), 6.79 (2H,
brd d, ArH), 3.43 (8H, brd, NCH.sub.2), 1.05 (12H, brd t,
CH.sub.3); .delta..sub.C (62.9 MHz; D.sub.2O) 12.3 (CH.sub.3), 56.2
(NCH.sub.2), 117.9 (ArC), 121.4 (ArC), 122.4 (ArC), 124.5 (ArC),
133.5 (ArC), 145.1 (ArC).
Example 10
Other Cognitive or CNS Disorders
[0795] Methods of treatment, prophylaxis, diagnosis or prognosis of
the present invention, utilising DAPTZ compounds in oxidised or
reduced form, may in any aspect be applied to any one or more of
the following diseases.
TABLE-US-00016 Diseases of protein aggregation Fibril Aggregating
subunit domain and/or size Protein Disease mutations (kDa)
Reference Neuro-degenerative disorders Prion protein Prion diseases
Inherited and 27 Prusiner (1998) sporadic forms (CJD, nvCJD, Fatal
PrP-27-30; many familial insomnia, mutations. Gerstmann-Straussler-
Scheinker syndrome, Kuru) Fibrillogenic Gasset et al. domains:
113-120, (1992) 178-191, 202-218. Tau protein Alzheimer's disease,
Inherited and 10-12 Wischik et al. Down's syndrome, sporadic forms
(1988) FTDP-17, CBD, post- encephalitic parkinsonism, Pick's
disease, parkinsonism with dementia complex of Guam Truncated tau
(tubulin-binding domain) 297-391. Mutations in tau Hutton et al. in
FTDP-17. (1998) Many mutations Czech et al. in presenilin (2000)
proteins. Amyloid Alzheimer's disease, Inherited and 4 Glenner
& .beta.-protein Down's syndrome sporadic forms Wong, (1984)
Amyloid .beta.- protein; 1-42(3). 11 mutations in Goate et al. APP
in rare (1991) families. Huntingtin Huntington's disease N-termini
of 40 DiFiglia et al. protein with (1997) expanded glutamine
repeats. Ataxins Spinocerebellar ataxias Proteins with Paulson et
al. (1, 2, 3, 7) (SCA1, 2, 3, 7) expanded (1999) glutamine repeats.
Atrophin Dentarubropallidoluysian Proteins with Paulson et al.
atrophy (DRPLA) expanded (1999) glutamine repeats. Androgen Spinal
and bulbar Proteins with Paulson et al. receptor muscular atrophy
expanded (1999) glutamine repeats. Neuroserpin Familial
encephalopathy Neuroserpin; 57 Davis et al. with neuronal inclusion
S49P, S52R. (1999) bodies (FENIB) .alpha.-Synuclein Parkinson's
disease, Inherited and 19 Spillantini et al. dementia with Lewy
sporadic forms (1998) bodies, multiple system atrophy A53T, A30P in
Polymeropoulos rare autosomal- et al. (1997) dominant PD families.
Cystatin C Hereditary cerebral Cystatin C less 12-13 Abrahamson et
angiopathy (Icelandic) 10 residues; al. (1992) L68Q. Superoxide
Amyotrophic lateral SOD1 mutations. Shibata et al. dismutase 1
sclerosis (1996)
REFERENCES FOR EXAMPLE 10
[0796] Abrahamson, M., Jonsdottir, S., Olafsson, I. & Grubb, A.
(1992) Hereditary cystatin C amyloid angiopathy identification of
the disease-causing mutation and specific diagnosis by polymerase
chain reaction based analysis. Human Genetics 89, 377-380. [0797]
Czech, C., Tremp, G. & Pradier, L. (2000) Presenilins and
Alzheimer's disease: biological functions and pathogenic
mechanisms. Progress in Neurobiology 60, 363-384. [0798] Davis, R.
L., Shrimpton, A. E., Holohan, P. D., Bradshaw, C., Feiglin, D.,
Collins, G. H., Sonderegger, P., Kinter, J., Becker, L. M.,
Lacbawan, F., Krasnewich, D., Muenke, M., Lawrence, D. A., Yerby,
M. S., Shaw, C.-M., Gooptu, B., Elliott, P. R., Finch, J. T.,
Carrell, R. W. & Lomas, D. A. (1999) Familial dementia caused
by polymerization of mutant neuroserpin. Nature 401, 376-379.
[0799] DiFiglia, M., Sapp, E., Chase, K. O., Davies, S. W., Bates,
G. P., Vonsattel, J. P. & Aronin, N. (1997) Aggregation of
huntingtin in neuronal intranuclear inclusions and dystrophic
neurites in brain. Science 277, 1990-1993. [0800] Gasset, M.,
Bladwin, M. A., Lloyd, D. H., abriel, J.-M., Holtzman, D. M.,
Cohen, F. E., Fletterick, R. & Prusiner, S. B. (1992) Predicted
a-helical region of the prion protein when synthesized as peptides
form amyloid. Proceedings of the National Academy of Sciences, USA
89, 10940-10944. [0801] Glenner, G. G. & Wong, C. W. (1984)
Alzheimer's disease: initial report of the purification and
characterisation of a novel cerebrovascular amyloid protein.
Biochemical and Biophysical Research Communications 120, 885-890.
[0802] Goate, A., Chartier-Harlin, M.-C., Mullan, M., Brown, J.,
Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N.,
James, L., Mant, R., Newton, P., Rooke, K., Rogues, P., Talbot, C.,
Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M.
& Hardy, J. (1991) Segregation of a missense mutation in the
amyloid precursor protein gene with familial Alzheimer's disease.
Nature 349, 704-706. [0803] Hutton, M., Lendon, C., Rizzu, P.,
Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S.,
Chakraverty, S., Isaacs, A., Grover, A., Hackett, J., Adamson, J.,
Lincoln, S., Dickson, D., Davies, P., Petersen, R. C., Stevens, M.,
de Graaf, E., Wauters, E., van Baren, J., Hillebrand, M., Joosse,
M., Kwon, J. M., Nowotny, P., Che, L. K., Norton, J., Morris, J.
C., Reed, L. A., Trojanowski, J. Q., Basun, H., Lannfelt, L.,
Neystat, M., Fahn, S., Dark, F., Tannenberg, T., Dodd, P. R.,
Hayward, N., Kwok, J. B. J., Schofield, P. R., Andreadis, A.,
Snowden, J., Craufurd, D., Neary, D., Owen, F., Oostra, B. A.,
Hardy, J., Goate, A., van Swieten, J., Mann, D., Lynch, T. &
Heutink, P. (1998) Association of missense and 5'-splice-site
mutations in tau with the inherited dementia FTDP-17. Nature 393,
702-705. [0804] Paulson, H. L. (1999) Human genetics '99:
trinucleotide repeats. American Journal of Human Genetics 64,
339-345. [0805] Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide,
S. E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J.,
Boyer, R., Stenroos, E. S., Chandrasekharappa, S., Athanassiadou,
A., Papaetropoulos, T., Johnson, W. G., Lazzarini, A. M., Duvoisin,
R. C., Di lorio, G., Golbe, L. I. & Nussbaum, R. L. (1997)
Mutation in the a-synuclein gene identified in families with
Parkinson's disease. Science 276, 2045-2047. [0806] Prusiner, S.
B., Scott, M. R., DeArmond, S. J. & Cohen, F. E. (1998) Prion
protein biology. Cell 93, 337-348. [0807] Shibata, N., Hirano, A.,
Kobayashi, M., Siddique, T., Deng, H. X., Hung, W. Y., Kato, T.
& Asayama, K. (1996) Intense superoxide dismutase-1
immunoreactivity in intracytoplasmic hyaline inclusions of familial
amyotrophic lateral sclerosis with posterior column involvement.
Journal of Neuropathology and Experimental Neurology 55, 481-490.
[0808] Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M.
& Goedert, M. (1998) a-Synuclein in filamentous inclusions of
Lewy bodies from Parkinson's disease and dementia with Lewy bodies.
Proceedings of the National Academy of Sciences, USA 95, 6469-6473.
[0809] Wischik, C. M., Novak, M., Thogersen, N. C., Edwards, P. C.,
Runswick, M. J., Jakes, R., Walker, J. E., Milstein, C., M., R.
& Klug, A. (1988) Isolation of a fragment of tau derived from
the core of the paired helical filament of Alzheimer's disease.
Proceedings of the National Academy of Sciences, USA 85,
4506-4510.
Example 11
Standard Dissolution Test
[0810] Title: Simulated Intestinal Fluid Dissolution for DAPTZ
containing capsules.
[0811] Performed by: Encap Drug Delivery, Units 4, 5 & 6,
Oakbank Park Way, Livingston, West Lothian, EH53 0TH, Scotland,
UK.
1. Purpose
[0812] This method is suitable for use as a Dissolution Test Method
for the purpose of providing data for the determination of %
dissolution over time of DAPTZ containing dosage units in simulated
Intestinal Fluid (SIF), as described in the USP
(http://www.usp.org) as dissolution media.
[0813] The method is exemplified with 30 mg, 60 mg and 100 mg MTC
capsules formulated in Gelucire 44/14 and employs the standard
USP<711>Dissolution, Apparatus2 (paddle and sinker). Where
relevant below, the MTC can be replaced by an alternative DAPTZ
compound at the appropriate loading and.
2. Method Conditions
2.1. Reagents
[0814] Water--Lab. grade or equivalent
[0815] Potassium Dihydrogen Orthophosphate--Lab. grade or
equivalent
[0816] Sodium Hydroxide--Lab. grade or equivalent
[0817] Pancreatin--USP Grade
[0818] Hydrochloric Acid--Lab. grade or equivalent
2.2. Safety
[0819] Reagents are poss. irritant and poss. harmful.
2.3. Dissolution Conditions
[0820] Dissolution Apparatus
[0821] Apparatus--USP<711>Dissolution, Apparatus2 (paddle and
sinker)
[0822] Sample--1 capsule placed in a sinker
[0823] Rotation rate--75 rpm
[0824] Temperature--37.degree. C..+-.0.5.degree. C.
[0825] Dissolution Medium--1000 ml Simulated Intestinal Fluid
[0826] Sampling Times--15, 30, 45, 60 minutes
[0827] Test duration--60 minutes
[0828] Sample size--5 ml (not replaced) (Do not filter)
[0829] UV Spectrophotometer Conditions
[0830] Determination wavelength--665 nm
[0831] Reference--Dilute SIF
[0832] Path Length--10 mm
[0833] Band Width--2.0 nm
2.4. Preparation of Simulated Intestinal Fluid (SIF)
[0834] For each litre required, dissolve 6.8 g of potassium
dihydrogen orthophosphate in 250 ml of water, mix and add 77 ml of
0.2N Sodium Hydroxide and 500 ml of water. Add 10.0 g of pancreatin
mix, USP, and adjust the resulting solution with either 0.2N Sodium
Hydroxide or 0.2N Hydrochloric acid to a pH of 6.8.+-.0.1. Dilute
with water to 1000 ml. This solution must be prepared fresh every
day.
2.5. Standard Solutions (Prepare in Duplicate)
[0835] Accurately weigh approximately 100 mg of MTC into a 100 ml
volumetric flask. Dissolve in 80 ml of 50/50 ethanol water with 15
mins sonication and then make to volume with 50/50 ethanol/water
and mix well (1000 .mu.g/ml). Transfer 5.0 ml of this solution to a
100 ml volumetric flask and make this flask to volume with SIF and
mix well (50 .mu.g/ml). Transfer 4.0 ml of this solution to a 100
ml volumetric flask and make this flask to volume with water and
mix well. (2.0 .mu.g/ml). This is the standard solution.
2.6. Dissolution Procedure
[0836] Add 1000 ml of Simulated Intestinal Fluid to each of the six
dissolution vessels. Insert the paddles at the correct rotation
speed and allow to equilibrate to 37.degree. C..+-.0.5.degree. C.
Place six individual capsules into stainless steel sinkers and add
one to each vessel noting the time.
[0837] At each of the specified times withdraw a 5 ml sample.
2.7 Preparation of Background Reference
[0838] Transfer 4.0 ml of SIF to a 100 ml volumetric flask and make
to volume with water and mix well. This solution is to be used as
the background reference in the UV Spectrophotometer.
2.8 Sample Preparation
[0839] For the 30 mg capsules transfer 3.0 ml of this solution to a
50 ml volumetric flask and make to volume with water and mix well
(1.8 .mu.g/ml).
[0840] For the 60 mg capsules transfer 3.0 ml of this solution to a
100 ml volumetric flask and make to volume with water and mix well
(1.8 .mu.g/ml).
[0841] For the 100 mg capsules transfer 1 ml of this solution to a
50 ml volumetric flask and make to volume with water and mix well
(2.0 .mu.g/ml). These are the sample solutions.
2.9. Procedure
[0842] Determine the standard and sample solutions on a UV
Spectrophotometer that has been turned on and allowed to warm to
operating temperature.
2.10 Standard Verification
[0843] Verify the mean response factors of two standard solutions.
Standard 2 must verify as 98-102% of standard 1.
2.11 Calculations
[0844] Conduct all calculations to 2 decimal places
[0845] Determine the MTC % release of each sample relative to the
reference standard using the appropriate equation:
% release for 100 mg capsule=Asam/Astd.times.Wstd/(100
mg).times.P.times.100
% release for 60 mg capsule=Asam/Astd.times.Wstd/(60
mg).times.2/3.times.P.times.100
% release for 30 mg capsule=Asam/Astd.times.Wstd/(30
mg).times.1/3.times.P.times.100
[0846] Asam is the MTC Absorbance for the individual sample at 665
nm
[0847] Astd is the mean MTC Absorbance of the two standards at 665
nm
[0848] Wstd is the mean weight of MTC standards used (mg)
[0849] P is the Purity of reference standard used, as a decimal (eg
0.999)
[0850] (Where the input material is used as a standard a correction
factor of 1 is applied for P)
[0851] Plot the MTC % Release against the dissolution time on one
graph where individual vessels are plotted separately.
[0852] Plot the mean MTC % Release, across all six vessels, against
the dissolution time on one graph.
[0853] Thus generally the following equation can be used.
% release for x mg
capsule=Asam/Astd.times.Wstd/(x).times.d.times.P.times.100
[0854] It will be appreciated by those skilled in the art that `d`
is the correction, if required, for dilution in sample preparation
as in step 2.8 above.
2.12 Standard test for Simulated Gastric Fluid (SGF)
[0855] This standard test is carried out as described above but
using SGF in place of SIF. SGF is prepared according to USP29 as
follows:
[0856] Gastric Fluid, Simulated, TS-Dissolve 2.0 g of sodium
chloride and 3.2 g of purified pepsin, that is derived from porcine
stomach mucosa, with an activity of 800 to 2500 units per mg of
protein, in 7.0 mL of hydrochloric acid and sufficient water to
make 1000 mL. [Pepsin activity is described in the Food Chemicals
Codex specifications under General Tests and Assays]. This test
solution has pH of about 1.2.
Example 12
Quantitative Models for the Progression and Treatment of
Alzheimer's Disease
[0857] The chemical process underlying Alzheimer's Disease is the
aggregation and truncation of tau proteins. In this Example, we use
kinetic models of the tau reaction pathway in order to describe the
progression of the disease and the effect of treatment, and to
compare the effectiveness of treatments which target different
parts of the pathway.
1. Formulating an Equilibrium Model
[0858] FIG. 37A shows the binding of a tau protein to an aggregate
of truncated tau proteins, followed by the truncation of the tau
protein to form a larger aggregate. Within the cell this reaction
is embedded in a larger pathway, with paths for the creation of new
tau proteins and for the clearance of aggregates.
[0859] FIG. 37B shows a natural model. Here, S denotes the amount
of soluble tau protein, and A the amount of aggregated truncated
tau. In order to produce a kinetic model, we need to specify rates.
It is known that the rate of aggregation of tau increases with both
the availability of S and the availability of A [Wischik, C. M.,
Edwards, P. C., Lai, R. Y. K., Roth, M. & Harrington, C. R.
(1996) Selective inhibition of Alzheimer disease-like tau
aggregation by phenothiazines. Proceedings of the National Academy
of Sciences, USA 93, 11213-11218]. It is natural to assume that
there is a feedback mechanism involved in the creation of S, and
thus that the rate of production of S depends on the amount of S
[Lai, R. Y. K., Gertz, H.-J., Wischik, D. J., Xuereb, J. H.,
Mukaetova-Ladinska, E. B., Harrington, C. R., Edwards, P. C., Mena,
R., Paykel, E. S., Brayne, C., Huppert, F. A., Roth, M. &
Wischik, C. M. (1995) Examination of phosphorylated tau protein as
a PHF-precursor at early stage Alzheimer's disease. Neurobiology of
Aging 16, 433-445.]. For the other pathways shown, we will make the
standard kinetic assumption that the rate of a reaction is
proportional to the amount of reagent.
[0860] This gives us the kinetic model shown in FIG. 37C. By this
picture we mean, for example, that if S(t) is the amount of soluble
tau protein at time t then
d/dtS(t)=.lamda.(S(t))-k.sub.S0S(t)-kA(t)S(t) [equation 1]
Timescales of Disease Progression and of Kinetics
[0861] A crucial aspect of this model is the timescale over which
Alzheimer's Disease progresses, and its relationship with the
timescale over which equations like equation 1 operate. It is our
position that the dynamics of the kinetic equations occur over
hours or days, and that the progression of the disease is a due to
the slow change of parameters like k.sub.A0 over the timescale of
years. A contrary position was adopted in Wischik et al. (1995),
namely that the timescale of the kinetics is measured in years, and
that the progression of the disease reflects the gradual increase
of A(t) as modelled by the kinetics.
[0862] There are two main pieces of evidence for the separation of
timescales. First, in vitro experiments [WO96/30766], in which
soluble tau is incubated with solid-phase truncated tau, show that
most of the soluble tau has bound within a matter of hours. The
second piece of evidence comes from in vivo experiments on
transgenic mice which express human truncated tau protein [WO
02/059150]. These mice slowly develop Alzheimer's disease tau
pathology over periods of months, as measured both by cognitive
tests and by neuropathological examination [Zabke, C., Dietze, S.,
Stamer, K., Rickard, J. E., Harrington, C. R., Theuring, F., Seng,
K. M. & Wischik, C. W. (2008) Early and advanced stages of tau
aggregation In transgenic mouse models. International Conference on
Alzheimer's Disease, Chicago, 26-31 Jul. 2008, P1-054]. When
treated with daily oral doses of MTC over a period of 17 days, the
Alzheimer's disease pathology was reduced [Harrington, C., Rickard,
J. E., Horsley, D., Harrington, K. A., Hindley, K. P., Riedel, G.,
Theuring, F., Seng, K. M. & Wischik, C. M. (2008)
Methylthioninium chloride (MTC) acts as a tau aggregation inhibitor
(TAI) in a cellular model and reverses tau pathology in transgenic
mice models of Alzheimer's disease. International Conference on
Alzheimer's Disease, Chicago, 26-31 Jul. 2008, O1-06-04]. Therefore
the timescale of the kinetics is of the order of days, while the
timescale of the progression of the disease is much longer,
measured in months for these mice.
[0863] Our mathematical technique must therefore be this: we
suppose that any patient has rate constants which depend on how
long he has had the disease, say k.sub.A0(a) etc. where a is the
number of years since onset; and we suppose that the resulting
levels of S and A are the equilibrium values of the dynamical
system. To be concrete, we need to solve equations like this
modified form of equation 1:
.lamda.(S)-k.sub.S0S-kAS=0. [equation 2]
[0864] We have omitted t, since we are not interested in the
dynamics of the system but only in the equilibrium behaviour. We
will sometimes write S(a) etc. to emphasize the dependence on the
values of the rate constants.
Accounting for the Creation of New Aggregates
[0865] The aggregation reaction (FIG. 37A) starts with one
aggregate molecule and finishes with one aggregate molecule, so it
describes the growth of existing aggregates and not the creation of
new aggregates. Likewise in the kinetic system (FIG. 37C), if we do
not model the creation of aggregates then the pool of A will
steadily decrease, meaning that the equilibrium solution is
A=0.
[0866] The simplest way to account for the creation of new
aggregates is by altering the stoichiometry of the aggregation
reaction. Specifically, we will assume the scheme shown in FIG. 37D
(though the actual values of n.sub.1 and n.sub.2 are unknown).
[0867] For example, if n.sub.1=2.3 and n.sub.2=1.87 then from 230
tau molecules and 100 aggregate molecules there are 87 new
aggregate molecules produced.
Summary of Model
[0868] We have proposed the dynamical system model shown in FIG.
37E.
[0869] The equations for the equilibrium state of this system
are:
.lamda.(S)=k.sub.S0S+n.sub.1kAS [equation 3]
n.sub.2kAS=kAS+k.sub.A0(.alpha.)A [equation 4]
[0870] In the remainder of this Example we describe several
experiments which let us quantify the rate constants and thus to
predict the effect of treatment.
2. Quantifying the Progression of Disease
[0871] Lai et al. (1995) studied a number of Alzheimer's patients
post-mortem and found a relationship between A and S:
S=f(A)=.alpha./A.sup..beta.1 [equation 5]
where .alpha.=2450 and .beta.=0.3459.
[0872] Mukaetova-Ladinska et al. (Mukaetova-Ladinska, E. B.,
Garcia-Siera, F., Hurt, J., Gertz, H. J., Xuereb, J. H., Hills, R.,
Brayne, C., Huppert, F. A., Paykel, E. S., McGee, M., Jakes, R.,
Honer, W. G., Harrington, C. R. & Wischik, C. M. (2000) Staging
of cytoskeletal and .beta.-amyloid changes in human isocortex
reveals biphasic synaptic protein response during progression of
Alzheimer's disease. American Journal of Pathology 157, 623-636)
studied a number of Alzheimer's patients pre- and post-mortem, and
found a relationship between PHF levels and the patient's Braak
stage B:
PHF=g(B)=Exp(.gamma.B/(.delta.-B))-1 [equation 1]
where .gamma.=4.8383 and .delta.=9.8156.
[0873] It is reasonable to assume that PHF levels are proportional
to levels of tau aggregates:
A=.epsilon.PHF [equation 2]
though .epsilon. is unknown.
[0874] Ohm et al. [Ohm, T. G., Muller, H., Braak, H. & Bohl, J.
(1995) Close-meshed prevalence rates of different stages as a tool
to uncover the rate of Alzheimer's disease-related neurofibrillary
changes. Neuroscience 64, 209-217] studied the distribution of
Braak stage within a population, and in the appendix we describe
how from his data we can obtain a relationship between mean Braak
stage B and the time a since the onset of dementia, in years:
B=h(a)= [equation 8]
[0875] Using these three relationships, we can rewrite the
equilibrium equations 3-4 to obtain:
.lamda.(S)=k.sub.S0S+n.sub.1kf.sup.-1(S)S [equation 9]
k.sub.A0(a)=(n.sub.2-1)kf(.epsilon.g(h(a))) [equation 10]
3. Quantifying the Effect of a Drug
[0876] WO 02/055720 describes a cell model for Alzheimer's disease,
and measurements demonstrating the effect of MTC on levels of A.
The cells have been genetically modified to produce soluble tau S
at a constant rate. On its own, this does not spontaneously form
aggregates, and so the cells have been further modified to produce
truncated tau T at a constant rate. We assume that the cells have a
normal mechanism for destroying T, and that the effect of the drug
is to open up a pathway by which A is dissolved and turns into T.
For simplicity, we assume that here S is only used in the
Alzheimer's pathway. We therefore have the kinetic model shown in
FIG. 37F.
[0877] We have written k.sub.AT(d) to emphasize that this rate
constant depends on the dose level d, and we will assume that
k.sub.AT(0)=0. We should strictly write k.sub.A0(a.sub.cell), where
a.sub.cell is the time in years since the onset of the disease for
these cells, though we will suppress this in our equations.
[0878] The equilibrium equations for this system are:
.lamda.=kASn.sub.1 [equation 11]
.mu.+k.sub.AT(d)A=T(k.sub.T0+k.sub.TA) [equation 12]
k.sub.TAT+n.sub.2kAS=kAS+A(k.sub.AT(d)+(k.sub.A0) [equation 13]
[0879] Using equations 11 and 12, we can eliminate S and T from
equation 12 to obtain:
A=[(n.sub.2-1)/n.sub.1.lamda.+k.sub.TA/(k.sub.T0+k.sub.TA).mu.]/[k.sub.A-
0+k.sub.AT(d)k.sub.T0/(k.sub.T0+k.sub.TA)]
[0880] Writing A(0) for the baseline level of aggregate tau, in the
absence of any drug, then:
A(0)=[(n.sub.2-1)/n.sub.1.lamda.+k.sub.TA/(k.sub.T0+k.sub.TA).mu.]/k.sub-
.A0
[0881] These two equations cancel conveniently, and tell us
that:
k.sub.AT(d)/k.sub.A0=(1+k.sub.TA/k.sub.T0)(A(0)/A(d)-1)
(We have written A(d) here to emphasize that the observed level of
aggregates A is a function of the dose d).
[0882] WO 02/055720 reports that:
A(d)/A(0)=g(d)=.zeta.d.sup..theta./(.eta..sup..theta.+d.sup..theta.)+1
equation 14]
where .zeta.=-1.0665, .eta.=51.735 and .theta.=1.3328.
4. Quantifying the Combined Effect
[0883] We can now ask: how to we expect the drug would alter the
progression of the disease? Our kinetic model is now that shown in
FIG. 37G, with equilibrium equations:
.lamda.(S)=k.sub.S0S+n.sub.1kAS [equation 15]
k.sub.AT(d)A=T(k.sub.T0+k.sub.TA) [equation 16]
k.sub.TAT+n.sub.2kAS=kAS+A(k.sub.AT(d)+k.sub.A0(a)) [equation
17]
[0884] We wish to solve these equations for A=A(a,d). To do this,
it is most convenient to use equation 16 to express T in terms of
A:
T=Ak.sub.AT(d)/k.sub.T0+k.sub.TA)
and then to substitute into 17 to find an expression for
S=S(a,d)
(n.sub.2-1)kS(a,d)=k.sub.A0(a)+k.sub.AT(d)k.sub.TO/(k.sub.T0+k.sub.TA)
and finally to use equations 15 and 9 to turn this into an
expression for A(a,d)
A(a,d)=C(S(a,d)) [equation 18]
[0885] The expression for S(a,d) can more usefully be written as a
ratio involving S(a.sub.0,0) where a.sub.0 is the time since the
onset of the disease at which treatment was begun. We shall also
substitute in the expressions we have obtained for k.sub.A0(a) and
k.sub.AT(d), to give:
S(a,d)/S(a.sub.0,0)=k.sub.A0(a)/k.sub.A0(a.sub.0)+k.sub.A0(a.sub.cell)/k-
.sub.A0(a.sub.0) (1/g(d)-1) [equation 19]
[0886] The formula for g(d) is given above in equation 14, the
formula for f is given in equation 8, and the formula for
k.sub.A0(a) is given in equation 9.
[0887] Interpretation of the result.
[0888] If we let d=0, equation 19 gives:
S(a,0)/S(a.sub.0,0)=k.sub.A0(a)/k.sub.A0(a.sub.0)
[0889] As a increases, the pathway by which aggregates are cleared
degenerates, and k.sub.A0(a) decreases towards 0; thus S(a,0)
decreases towards 0 and, according to equation 18, A increases to
infinity. By treating with the drug at some fixed dose, we prevent
S from decreasing below a certain threshold:
S.sub.thresh=k.sub.A0(a.sub.cell)/k.sub.A0(a.sub.0)(1/g(d)-1)
which means that we prevent A from increasing above a certain
threshold f.sup.-1(S.sub.thresh). In words, this treatment does not
merely retard the progression of the disease, it stops it.
5. An Alternative Treatment Model
[0890] It has been suggested that one might treat Alzheimer's
disease inter alia by inhibiting the tau-tau binding reaction
(Wischik, C. M., Edwards, P. C., Lai, R. Y. K., Roth, M. &
Harrington, C. R. (1996) Selective inhibition of Alzheimer
disease-like tau aggregation by phenothiazines. Proceedings of the
National Academy of Sciences, USA 93, 11213-11218). What effect
would this have on the progression of the disease? Consider the
kinetic model shown in FIG. 37H where k(d) is the value of the rate
constant, after the reaction has been inhibited by this putative
drug at dose d. The equilibrium equations are:
.lamda.(S)=k.sub.S0S+n.sub.1k(d)AS
n.sub.2k(d)AS=k(d)AS+Ak.sub.A0(a)
[0891] Solving these, and substituting in equation 8, we
obtain:
S(a,d)/S(a.sub.0,0)=[k.sub.A0(a)/k.sub.A0(a.sub.0)]/[k(d)/k(0)]
A(a,d)=f.sup.-1(S(a,d))/[k(d)/k(0)]
[0892] It can be seen that the level of S(a,d) decreases to 0 as
time a increases, for any fixed dose d. Therefore the level of A
increases to infinity. In words, a treatment based purely on
inhibition of the tau-tau binding reaction would retard the
progression of the disease, but it could not halt it.
6. Numerical Results
[0893] FIG. 37I illustrates these results numerically. The left
plot shows the effect of a drug which creates a new pathway
A.fwdarw.T, as described in Section 3; the left plot shows the
effect of a drug which inhibits the pathway S+A.fwdarw.A, as
described in Section Error!Reference source not found. Rather than
plotting the level of tau aggregates A, we have plotted MMSE, using
the relationship between MMSE and Braak stage B derived from data
in Ohm et al. (1995).
MMSE=.sigma.(.tau.-B)/(.rho.-B)
where .sigma.=56.2204, .tau.=6.5969 and .rho.=11.599, together with
the relationships in equations 6 and 7, and setting .epsilon.=1. We
plot this as a function of number of years since the beginning of
treatment, for a patient who started treatment at MMSE=15. The
dotted line shows the deterioration of MMSE with no treatment; the
other lines show the effect of treatment at various dose levels.
The dose levels we are illustrating here are (for the left plot)
d=25, 50 and 90; and (for the right plot) k(d)/k(0)=45%, 20%,
7%.
7. Implications for Clearance of Tau Aggregates for Disease
Progression
[0894] These figures (FIG. 37I) illustrate what we have already
explained algebraically, namely that inhibiting tau-tau aggregation
can only retard the progression of the disease, whereas it can be
halted by opening a new pathway for dissolution of aggregates. This
can be depicted schematically in FIG. 39. Tau aggregation can be
prevented by affecting two sites: firstly by inhibiting the input
of tau into the cycle of aggregation and secondly by enhancing the
clearance of aggregates from the aggregation cycle (FIG. 39). The
level of aggregated tau or paired helical filaments progresses
steadily with advancing age. If the input of tau is prevented, then
the level of PHFs will decrease to a certain level, predicted by
Braak staging, after which time the rate of progression will
continue as before. Only when the clearance of aggregated tau is
enhanced will their levels of tau begin to decrease over time (FIG.
39). In such circumstances, a drug having such an effect can be
said to be disease-modifying. It has been discussed by Wischik et
al. (Wischik, C. M., Lai, R. Y. K. & Harrington, C. R. (1997)
Modelling prion-like processing of tau protein in Alzheimer's
disease for pharmaceutical development. In Microtubule-Associated
Proteins: Modifications in Disease. (Eds. J. Avila, R. Brandt,
& K. S. Kosik) Harwood Academic Publishers, Amsterdam, 185-241)
that tau aggregation can be seeded by proteins arising from
age-related mitochondrial turnover (e.g. core protein 2 of complex
III, porin and ATP synthetase subunit 9). These aggregates of tau
can either assemble into PHFs and/or enter the endosomal-lysosomal
clearance pathway, adding to the congestion of this pathway with
advancing age (FIG. 40). Enhanced clearance of tau aggregates from
this pathway that will decrease the metabolic burden within the
neuron. This Example demonstrates how this could halt the
progression of the disease, rather than just retard its
progression.
* * * * *
References